1-methyl-2-pyrrolidone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral route:

Two reproduction toxicity studies in line with the requirements of OECD 416 were performed in Sprague Dawley and Wistar rats using dietary dose levels of 0, 50, 160 and 500/350 mg/kg bw/day.

In both studies, the high dose level was reduced to 350 mg/kg bw/day due to severe pup mortality in the first litter. Both rat strains were very similar with respect to the observed findings. All F0 and F1 parental rats proved to be fertile at least after one of the two mating intervals. The NOAEL for reproductive performance/fertility was 350 mg/kg bw/day in both strains (BASF SE, 1999 and Huntingdon Life Sci, 1999).

At far higher doses in another more supportive study focusing soley on male reproductive performance, subchronic NMP exposure of male Wistar rats by gavage resulted in gonadotoxic effects at the high dose level of 1000 mg/kg bw/day, while NMP did not significantly affect fertility and spermatogenesis at the low and mid dose levels of 100 and 300 mg/kg bw/day, respectively. It has to be noted that there are also other toxic effects at that dose (Sitarek and Stetkiewicz, 2008).

Beyond this no effects in testes were seen at histopathology in dietary repeated dose toxicity studies at the highest dose (18000 ppm) corresponding to 1060 mg/kg (Haskell, 1995).

Inhalative route:

In a two-generation reproduction study in rats 10 males and 20 females per dose level were exposed whole body to 0, 10 ppm (41 mg/m3), 51 ppm (206 mg/m3) or 116 ppm (478 mg/m3) of NMP vapour (Solomon et al., 1995). Sprague-Dawley rats were exposed for 6h/day, 7 days/week, for a minimum of 14 weeks.

There were no effects observed for the reproducibility or the reproductive organs. For maternal toxicity a NOAEC of 51 ppm (206 mg/m3) is proposed. However, as the only effect in maternal animals was the reduced sensitivity to sound in parental animals before mating, this effect is considered to be a systemic toxicity effect.

The developmental LOAEC is 116 ppm (478 mg/m3) as effects to the pup body weight in the developmental part of the study (Solomon et al., 1995) and reduced body weight gain for the F1 generation is described in the study.

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-06-04 to 1998-06-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

May 1983

Deviations:

yes

Remarks:

Due to excessive F1 pup mortality, the high dose was reduced from 500 mg/kg bw/day to 350 mg/kg bw/day after day 126 (18 weeks of treatment). Extra observations, such as percent motility, sperm count/morphology and ophthalmoscopic examinations.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, Ludwigshafen, Germany; tank No. 32, 26-02-97
- Analytical purity: 99.9 %
- Expiration date of the lot/batch: 27 july 1998
- Purity test date: 15 April 1997

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under N2, in brown glass containers

FORM AS APPLIED IN THE TEST: mixed with commercial rat diets

Species:

rat

Strain:

Wistar

Remarks:

Chbb = THOM (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl Thomae, Biberach an der Riss, Germany
- Details on animals: females, nulliparous and non-pregnant; male and female animals were derived from different litters (written statement of the breeder)
- Age at study initiation: (P) 28 ± 1 day (at arrival) plus about 15 days (acclimation)
- Weight at study initiation: (P) Males: 196.3 g (167.0 - 222.0 g); Females: 150.5 g (130.7 - 165.7 g)
- Housing: individually in type DK III stainless steel wire mesh cages (Becker & Co., Castrop Rauxel, Germany).
Exception: from day 18 of gestation until day 14 after birth the pregnant animals and their litters were housed in Markrolon type M III cages (Becker & Co., Castrop Rauxel, Germany)
- Diet: Kliba maintenance diet rat/mouse/hamster (Klingentalmühle AG, new company name Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: from water bottles, ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours

IN-LIFE DATES: From: 04 June 1997; To: 19 June 1998

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: test diets were prepared at intervals which guaranteed that the concentration of test substance in the diet remained stable throughout the feeding period
- Mixing appropriate amounts with: control diet

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 days (three oestrous cycles)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually, type III cage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of NMP in the food over a period of up to 32 days at room temperature was proven prior to dosing.

The homogeneous distribution of the test substance in the diet was examined before the beginning of the administration period. Samples of each concentration prepared were drawn for concentration control analyses at the start of the administration period, thereafter about 1, 2, 3, 7, 10, 12, 15, 21, 22, 25, 27, 33, 39, 42, 48, 50 weeks after the beginning and at termination of the in life phase.

Duration of treatment / exposure:

Exposure period:
F0: 10 weeks premating, mating, gestation/lactation and rest period of F1a and F1b offspring
F1: after weaning during 10 weeks premating, mating, gestation/lactation and rest period F2a/F2b offspring
F2: until weaning
Premating exposure period (males): 10 weeks Premating exposure period (females): 10 weeks
Duration of test: approx. 54 weeks

Frequency of treatment:

continuous (7d/w)

Details on study schedule:

- Number of generation studies: 2
- F1 parental animals not mated until 70 days after selected from the F1 litters.
- Selection of parents from F1 generation was performed before weaning.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

160 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

After day 126 (18 weeks of treatment) the high-dose level was reduced from 500 to 350 mg/kg BW/day due to excessive mortality of the F1a pups; i.e. in the F0 parental males (from study week 17 onwards), in the F0 parental females (after weaning of the F1a pups), in the F1b pups, the F1 parents and their progeny (F2a and F2b litters) .

No. of animals per sex per dose:

25 animals

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
In a two-generation reproduction toxicity study (with two litters in each parental generation) conducted by EXXON Biomedical Sciences Inc. in 1991 (unpublished study), NMP was administered to Charles River CrL:CdBR (Sprague-Dawley) VAF/Plus rats in the diet at dose levels of about 0, 50, 160 and 500 mg/kg bw/day. In the Exxon study decreased pup survival and decreased reproduction and fertility (second parental generation only) were observed together with parental toxicity at the highest dose level (500 mg/kg bw/day) tested . Adverse effects on fertility/reproduction parameters observed at the lower doses were considered by the authors of this study "to be spurious findings (...) and not treatment-related". However, the US EPA concluded that the reductions in male fertility and female fecundity observed at the low- and intermediate-dose levels were biologically (although not statistically) significant and that a NOAEL was not achieved in this study.

On request of the Sponsor, dietary concentrations were selected to achieve nominal dose levels of 0, 50, 160 and 500 mg/kg bw/day for the present two-generation reproduction toxicity study in Wistar rats. As excessive pup mortality was observed in the F1a litters in this study at 500 mg/kg bw/day, it was decided to reduce the high dose level to 350 mg/kg bw/day after weaning of the surviving F1a pups for the remainder of the study; i.e. in the F0 parental males (from week 17 onwards), in the F0 parental females (after weaning of the F1a pups), in the F1b pups, the F1 parents and their progeny (F2a and F2b litters) .

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:
In general, the body weight of the parental animals was determined on the first day of dosing and thereafter once a week at the same time of the day (in the morning). However, during gestation and lactation F0/F1 females were weighed on days 0, 7, 14 and 20 of gestation, and on days 1, 4, 7, 14 and 21 after birth. The body weight change of the animals was calculated from these results.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption of the F0 and F1 parents was determined regularly during premating (once weekly, in general over a period of 6 days each), and additionally during gestation and lactation periods of the F0 and F1 females.

OTHER: At least once daily a check was made for dead or moribund animals.

Oestrous cyclicity (parental animals):

Estrous cycle data were evaluated for F0 and F1 generation females over a three week period prior to mating for the first pregnancies and throughout the following mating period (up to 14 days) until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

Sperm parameters (parental animals):

Different sperm parameters (motility, sperm head count, morphology) were assessed in all F0 and F1 generation males at scheduled sacrifice or shortly thereafter.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed.
The F1a, F1b, F2a and F2b pups were sexed, and weighed on the day after birth and on days 4, 7, 14 and 21 post partum. Their viability was recorded. Sexual maturation (day of preputial separation/vaginal opening) of all pups selected to become F1 parental generation animals was determined.
Standardization of litters was not performed in litters with < 10 pups. Moreover, due to the excessive pup mortality at the high concentration (i.e. 500 mg/kg body weight/day) in the first litter (F1a) of the F0 parental rats, standardization was not performed in this test group.

Postmortem examinations (parental animals):

All F0 and F1 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination, special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial follicles, growing follicle and antral follicles in the ovaries was performed for all control and high dose F0 and F1 parental females. Moreover, blood was collected from all F0 and F1 generation parental animals at sacrifice and frozen for future possible analyses.

Postmortem examinations (offspring):

After scheduled sacrifice brain, spleen and thymus of 3 pups/sex and litter from the F1a, F1 b (not selected to become F1 parents), F2a and F2b pups each were weighed. Normally, the first 3 male and first 3 female pups/litter were taken for these determinations. Pup body weights routinely taken during the in-life phase on day 21 p.p. were used to calculate relative pup organ weights .
All pups were examined macroscopically at necropsy (including weight determinations of brain, spleen and thymus in 3 pups/sex/litter); certain pupswere additionally inspected for further skeletal findings. 

Statistics:

See table 1 in the section: Any other information on materials and methods incl. tables

Reproductive indices:

The following indices were calculated:
Male: male mating index, male fertility index
Female: female mating index, female fertility index, gestation index, live birth index

Offspring viability indices:

Viability index, lactation index

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In all substance-treated groups a dose-related discoloration of urine in the bedding was observed.
This finding was already observed in several other toxicity studies in different species and is assessed to be a sign of systemic availability of the test substance, not an adverse effect.
No further clinical signs which might be attributed to the test substance were detected in male or female F0 generation parental animals. There were no disturbances of the general behavior noted in any of the F0 parental animals.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled mortalities in any of the F0 generation parental animals in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The high dose F0 dams showed impairments in body weight/body weight gain during gestation and lactation of F1a litters (500 mg/kg bw/day) and during the gestation period of the second litter (F1b - 350 mg/kg bw/day) with concurrent impairments in food consumption; however, the high number of complete F1a litter losses at 500 mg/kg bw/day and the impaired high dose F1a and F1b pup body weights/body weight gains may at least partly account for these impairments. Moreover, slight impairments in F1 female body weight data at 350 mg/kg bw/day during premating and F2a gestation occurred.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was impaired as discussed for the effects observed in body weights.

Food efficiency:

no effects observed

Description (incidence and severity):

The measured intakes of NMP by the different test groups (calculated on the basis of interpolated mean body weights) generally correlated well with the desired target doses.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The increased incidence of focal cystic tubular dilation noted in the F0 and F1 generation high dose males and the focal papillary calcification in F1 high dose females are indicative for a possible marginal toxic effect of NMP on the kidney.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The administration of NMP had no adverse effect on estrous cycle data of the F0 parental females.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There occurred no treatment-related effects in the different sperm parameters examined at or after the sacrifice of parental males.

Reproductive performance:

no effects observed

Description (incidence and severity):

All F0 and F1 parental rats proved to be fertile at least after one of the two mating intervals each. The observable differences between the groups concerning the different reproductive parameters are regarded to be incidental in nature and are not of toxicological or biological concern.

See table 2 for more detailed information.

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Critical effects observed:

not specified

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased mean relative kidney weights in males and females

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

increased of focal cycstic tubular dilation in the kidneys of F1 males and of focal calcification in the renal papilla of F1 females

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

Dose descriptor:

LOAEL

Effect level:

350 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no apparent clinical signs of toxicity in offspring.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The administration of 500 mg NMP/kg bw/day led to a very high perinatal pup mortality rate (F1a). After decreasing the NMP dose from 500 to 350 mg/kg bw/day pup survival was still affected, but much less pronounced.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Impairments in pup body weight data occurred in the high dose progeny of the F0 and F1 parental rats (i.e. F1a, F1b, F2a and F2a litters).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between the substance-treated rats and the concurrent controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At high dose, organ weight changes secondary to the body weight impairments were noted in the progeny of F0 and F1 parental rats (i.e. F1a, F1b, F2a and F2b litters).

Gross pathological findings:

no effects observed

Description (incidence and severity):

The differences between the groups were regarded to be spontaneous in nature and not associated with the treatment of the animals.

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Generation:

F1a

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Generation:

F1b

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Generation:

F1b

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

350 mg/kg bw/day
- slightly increased pup mortality in both litters (viability index: F2a : 91 % vs. 96% in the control group, F2b: 89% vs. 96% in the control group; lactation index: F2a: unaffected, F2b : 94% vs. 100% in the control group)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

350 mg/kg bw/day
- lower mean body weights in both pup generations until weaning (about 7% below control on day 21 p.p.) and impaired body weight gains in these pups from day 4 p.p. up to weaning (about 6% less weight gain than in the controls)

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

350 mg/kg bw/day
- marginally lower absolute brain (about 2% below control) and spleen (about 7% below control) weights in both pup generations
- increased relative brain weight (about 5% above controls) in the F2a pups

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F2a

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Generation:

F2a

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Generation:

F2b

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Generation:

F2b

Effect level:

350 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

350 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Table 2: Maternal detailed results.

RELEVANT PARENTAL DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	50	160	500/350
Food Consumption
F0 males	-	-	-	-
F0 females	-	-	-	trans. reduced *
F1 males	-	-	-	-
F1 females	-	-	-	-
Body Weight
F0 males	-	-	-	-
F0 females	-	-	-	trans. reduced *
F1 males	-	-	-	-
F1 females	-	-	-	trans. reduced *
Body Weight Gain
F0 males	-	-	-	-
F0 females	-	-	-	trans. reduced *
F1 males	-	-	-	-
F1 females	-	-	-	trans. reduced *
Relative Kidney Weights
F0 males	0.62	0.63	0.66*	0.66*
F0 females	0.73	0.72	0.72	0.73
F1 males	0.61	0.63	0.64	0.66**
F1 females	0.75	0.75	0.77	0.78*
Histopathology
Kidney: Number and graded severity of focal cystic tubular dilation
F0 males	-	-	-	increased
F0 females	-	-	-	-
F1 males	-	-	-	increased
F1 females	-	-	-	-
Kidney: Number of focal calcification in the renal papilla
F0 males	-	-	-	-
F0 females	-	-	-	-
F1 males	-	-	-	-
F1 females	-	-	-	increased
-: no effect, trans. reduced: transiently reduced, *: p<0.05, ** p<0.01

Table 3: Indices for the maternal fertility and developmental results. 

RELEVANT REPRODUCTIVE PERFORMANCE DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	50	160	500/350
Mating Index [%]
F0 males/females (F1a)	100/100	100/100	100/100	100/100
F0 males/females (F1b)	100/100	100/100	100/100	100/100
F1 males/females (F2a)	100/100	100/100	100/100	100/100
F1 males/females (F2b)	100/100	100/100	100/100	100/100
Fertility Index [%]
F0 males/females (F1a)	100/100	100/100	100/100	100/100
F0 males/females (F1b)	96/96	100/100	100/100	100/100
F1 males/females (F2a)	100/100	96/96	96/96	88/88
F1 males/females (F2b)	100/100	100/100	100/100	100/100
Gestation Index [%]
F1a litter	100	100	100	100
F1b litter	100	100	100	100
F2a litter	100	100	100	95
F2b litter	100	100	100	96
Live Birth Index [%]
F1a litter	98	97	98	92**
F1b litter	100	97	98	98
F2a litter	98	99	99	98
F2b litter	97	98	99	98
* p<0.05, ** p<0.01, Cochran-Armitage trend test or Fisher’s Exact test

 

Table 4: Results and indices of the pup developmental performance.

RELEVANT DEVELOPMENTAL DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	50	160	500/350
Pups Delivered [mean]
F1a pups	13.1	13.2	13.6	12.3
F1b pups	14.0	13.6	13.1	11.0*
F2a pups	13.6	13.0	13.0	11.3
F2b pups	13.9	13.3	13.6	13.3
Viability Index [%, day 0-4 p.p.]
F1a pups	96	92*	97	18**
F1b pups	92	94	96	92
F2a pups	96	95	97	91*
F2b pups	96	95	98	89**
Lactation Index [%, day 4-21 p.p.]
F1a pups	100	98	99	88
F1b pups	100	98	100	94**
F2a pups	100	99	98	99
F2b pups	100	100	99	94**
Pups Body Weight Gain
F1a pups	-	-	-	reduced **
F1b pups	-	-	-	reduced **
F2a pups	-	-	-	reduced */**
F2b pups	-	-	-	reduced */**
Sexual Maturation
Preputial Separation	-	-	-	-
Vaginal Opening	-	-	-	-
-: no effect, *: p<0.05, ** p<0.01, p.p.: post partum

 

The stability of NMP in the food over a period of up to 32 days at room temperature was proven prior to dosing.The correctness of the homogeneous distribution in the diet and the dietary concentrations were proven. The measured intakes of NMP by the different test groups correlated generally well with the desired target doses.

 

Conclusions:

In a two-generation reproduction toxicity studies in Wistar rats, NMP had no adverse effects on reproductive performance or fertility of the F0 or F1 parental animals of all substance-treated groups. All F0 and F1 parental rats proved to be fertile at least after one of the two mating intervals (F0 parents: F1a and F1b; F1 parents: F2a and F2b) as demonstrated by the clinical and histopathological examinations. There were signs of systemic toxicity in each of the high dose groups at 500 mg/kg bw/day and also after reduction to 350 mg/kg bw/day. Parental toxicity consisted of reduced body weight gain and food intake as well as kidney findings in form of impaired organ weight and histopathological findings. Developmental toxicity was evidenced by increased pup mortality and reduced body weight gain, including corresponding effects in the investigated organs, in pups treated at 500/350 mg/kg bw/day.

Executive summary:

In a two-generation reproduction toxicity study, groups of 25 Wistar rats per sex were given NMP via the diet at initial dose levels of 0, 50, 160 or 500 mg/kg bw/day over a 10 -week premating period and throughout the mating, gestation, lactation and a rest period between pregnancies (BASF SE, 1999). The concentrations in the diet were adjusted regularly in respect to the actual body weight gain. Due to severe pup mortality in the first litter (F1a), the highest dose level was reduced to 350 mg/kg bw/day for the further course of the study. Each generation gave birth to two litters. The parental animals for the second generation were selected from pups of the second litter (F1b).

NMP had no adverse effects on reproductive performance or fertility of the F0 or F1 parental animals of all substance-treated groups. All F0 and F1 parental rats proved to be fertile at least after one of the two mating intervals (F0 parents: F1a and F1b; F1 parents: F2a and F2b) as demonstrated by the clinical and histopathological examinations. There were signs of systemic toxicity in each of the high dose groups at 500 mg/kg bw/day and also after reduction to 350 mg/kg bw/day. Parental toxicity consisted of reduced body weight gain and food intake as well as kidney findings in form of impaired organ weight and histopathological findings. Developmental toxicity was evidenced by increased pup mortality and reduced body weight gain, including corresponding effects in the investigated organs, in pups treated at 500/350 mg/kg bw/day.

Thus, the NOAEL for reproductive performance/fertility was 350 mg/kg bw/day. The NOAEL for systemic (parental) and developmental toxicity was 160 mg/kg bw/day.

This study is acceptable and satisfies the guideline requirement for a 2-generation reproductive study (OPPTS 870.3800; OECD 416) in rats.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-06-13 to 1998-07-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

May 1983

Deviations:

yes

Remarks:

Due to excessive F1 pup mortality, the high dose was reduced from 500 mg/kg bw/day to 350 mg/kg bw/day after day 126 (18 weeks of treatment). Extra observations, such as percent motility, sperm count/morphology and ophthalmoscopic examinations.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, Ludwigshafen, Germany; tank No. 32 26-02-97
- Analytical purity: considered 100 %
- Date received: 18 March 1997

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: stable in the diet for 32 days at ambient temperature as tested by the sponsor

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: mixed with commercial rodent diets in three different mixing steps
- Preliminary purification step (premix): manually mixing within a porcelain bowl

FORM AS APPLIED IN THE TEST: mixed with rodent diet

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD BR

Details on species / strain selection:

Historically, this strain has been the rat strain of choice for this type of study and a large database of control data are available to permit easy assessment and interpretation of results.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Portage, Michigan, USA
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: (P) Males: 204 - 258 g; Females: 129 - 169 g
- Housing: single, suspended, stainless-steel cage with wire mesh fronts and floors except mating, gestation and lactation. During mating one male and one female were cohabitated nightly. On gestation day 18, females were transferred to clear plastic "shoe-box type" solid cages with bedding material ("Bed-O-Cobs" corn cob bedding, Andersons, Maumee, Ohio, USA). During lactation dams and litter were housed together until weaning on day 21 of lactation.
- Diet: commercial diet (certified rodent diet No. 5002, PMI Feeds, St. Louis, MO, USA), ad libitum
- Water: automated watering system (Elizabethtown Water Company, Westfield, New Jersey, USA), ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): about 18 - 28 (above 24 on three occassions)
- Humidity (%): 26 - 82
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours

IN-LIFE DATES: From: 10 June 1997 To: 22 July 1998

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly (every 6 days)
- Mixing appropriate amounts with: control diet
- Storage temperature of food: room temperature

Details on mating procedure:

- M/F ratio per cage: 1:1 (in male's cage)
- Length of cohabitation: 14 days (three oestrous cycles).
- Proof of pregnancy: sperm seen in vaginal smear and/or presence of a copulatory plug in the vagina referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually for the remainder of gestation.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: housed individually for the remainder of gestation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of NMP in the food over a period of up to 32 days at room temperature was proven prior to dosing.

At initiation of the study, mock diet (test material and feed at the desired concentration) were prepared at the lowest and highest anticipated concentrations for use on study. Three samples each from the top, middle and bottom portion of each mix were taken for analysis.

All three test dietary levels for each sex were assayed for the following weeks: 1-4, 8, 11, 13, 16-18, 21, 23, 26, 29, 35, 41, 44, 51 and 54.

Duration of treatment / exposure:

Exposure period: F0: 10 weeks premating, mating, gestation/lactation and rest period of F1a and F1b offspring, F1: after weaning during 10 weeks premating, mating, gestation/lactation and rest period F2a/F2b offspring, F2: until weaning
Premating exposure period (males): 10 weeks
Premating exposure period (females): 10 weeks
Duration of test: approx. 58 weeks, from receipt of F0 to the sacrifice of the F1 parental generation and F2b pups

Frequency of treatment:

continuous (7d/week)

Details on study schedule:

- Number of generation studies: 2
- F1 parental animals were 114 days of age at initiation of mating

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

160 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

After day 126 (18 weeks of treatment) the high-dose level was reduced from 500 to 350 mg/kg BW/day due to excessive F1 pup mortality.

No. of animals per sex per dose:

30

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
In a two-generation reproduction toxicity study (with two litters in each parental generation) previously conducted by EXXON Biomedical Sciences Inc. (1991), NMP was administered to Charles River CrL :CdBR (Sprague-Dawley) VAF/Plus rats in the diet at dose levels of about 0, 50, 160 and 500 mg/kg bw/day. In the Exxon study decreased pup survival and decreased reproduction and fertility (second parental generation only) were observed together with parental toxicity at the highest dose level (500 mg/kg bw/day) tested . Adverse effects on fertility/reproduction parameters observed at the lower doses were considered by the authors of this study "to be spurious findings (...) and not treatment-related". However, the US EPA concluded that the reductions in male fertility and femal fecundity observed at the low- and intermediate-dose levels were biologically (although not statistically) significant and that a NOAEL was not achieved in this study.

On request of the Sponsor, dietary concentrations were selected to achieve nominal dose levels of 0, 50, 160, and 500 mg/kg bw/day for the P1 generation animals. Excessive pup mortality was observed in the F1a litters in this study at 500 mg/kg bw/day dose group, therefore, it was decided to reduce the dose level to 350 mg/kg bw/day beginning for both male and female rats on study day 126.

The detailed experimental design is given in table 1.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, exception: in F0 and F1 females, body weights were recorded at regular intervals during the gestation and lactation periods until sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule: weekly, exception: in F0 and F1 females, body weights were recorded at regular intervals during the gestation and lactation periods until sacrifice.

Oestrous cyclicity (parental animals):

Estrous cyclicity was evaluated for F0 and F1 generation female rats over a three-week period prior to the first cohabitation.

Sperm parameters (parental animals):

Parameters examined in [P/F1] male parental generations:
sperm motility, a count of homogenization-resistant testicular sperm, a count of caudal epididymal sperm and sperm morphology (cauda epididymal sperm

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

Postmortem examinations (parental animals):

All parental generation animals (F0 and F1) were provided to a gross post mortem examination. Reproductive tissues and selected organs were collected, weighed and preserved. Microscopic evaluations of reproductive tissues and gross lesions were performed for all F0 and F1 parental generation animals in the control and high dose groups. A quantitative assessment of primordial follicles in the ovaries was performed for all F0 and F1 parental generation animals in the control and high dose groups.

Postmortem examinations (offspring):

F1b pups not selected to become the F1 parental generation and all F1a, F2a and F2b pups were sacrificed at weaning on day 21 of lactation. These pups were given a macroscopic post mortem examination, and the brain, spleen and thymus were weighed. Only gross lesions from these animals were saved, all other tissues were discarded.

Statistics:

Bartlett's test followed by ANOVA and Dunnett's test or Kruskal-Wallis  test Fisher's exact test with Bonferroni correction.

Reproductive indices:

The following indices were calculated:
Male: male mating index, male fertility index.
Female: female mating index, female fertility index, gestation index, live birth index

Offspring viability indices:

Viability index, lactation index

Clinical signs:

no effects observed

Description (incidence and severity):

No significant clinical observations were seen in any NMP treatment groups.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The treatment-related effects were confined to the high dose females where there was a decrease in body weights at the end of gestation and throughout lactation.
After reduction of the high dose level from 500 to 350 mg/kg bw/day due to pup mortality, no effects were noted on body weight data and food consumption.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The treatment-related effects were confined to the high dose females where there was a decreased feed consumption during lactation
After reduction of the high dose level from 500 to 350 mg/kg bw/day due to pup mortality, no effects were noted on body weight data and food consumption.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopical findings.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

REPRODUCTIVE FUNCTION: ESTROUS CYCLE, SPERM MEASURES, REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no effect on reproductive performance or fertility as assessed by mating, gestation and delivery data as well as by sperm analysis and histopathology including ovarian follicle count

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no effects observed

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 350 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was a greater incidence of pups being pale, emaciated, cold to touch and containing no milk in the stomach at 500 mg/kg bw/day (F1a pups). These observations in the high dose group were a resut of treatment with NMP and correlates with the significant increase in the number of dams with pups who did not survive lactation. There were no treatment-related observations in the F1b, F2a and F2b pups.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The F1a pups exposed to 500 mg/kg bw/day had a decrease in mean litter size and pup survival as well as an increase of stillporn pups for dams exposed to 500 mg/kg bw/day.
Due to pup mortality the dose level of 500 mg/kg bw/day was reduced to 350 mg/kg bw/day for the remainder of the study and no effect was observed in the F1b pups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a decrease in body weight gain in F1a pups during lactation at 500 mg/kg bw/day. No effect was observed in the F1b pups treated at 350 mg/kg bw.
However, the F2a pups had initially reduced body weights (day 1 p.p. only) and the F2b pups had reduced body weights at the high dose of 500 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

No treatment-relaed effects were noted on sexual maturation in form of preputial separation and vaginal opening.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no effects on pup organ weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on pup morphology including malformations.

Histopathological findings:

no effects observed

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Generation:

F1a

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Generation:

F1b

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Generation:

F1b

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

organ weights and organ / body weight ratios

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no NMP treatment related clinical findings in the F2b pups during lactation.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The F2b pups at the 350 mg/kg BW/day NMP dose level had a decrease in the number of pups surviving lactation and a decrease in pup body weights.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean pup body weights evaluated by sex and for both sexes combined were significantly decreased for Lactation Day 1 for the 350 mg/kg BW/day treated pups.
For the remainder of lactation (Lactation Days 4-21), pup body weight in the 350 mg/kg BW/day NMP treated pup body weight was comparable to the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The F2b male pups exposed to 160 mg/kg bw/day and the F2b female pups exposed to 350 mg/kg bw/day had significant decreases in mean brain weights as a result of decreases in fetal body weight.
The change in mean brain weight was not considered NMP treatment-related since there were not corresponding changes in the other NMP treatment groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no NMP treatment-related effects on the F2a or F2b pups during the macroscopic postmortem examination on Lactation Day 21.

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F2a

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Generation:

F2a

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Generation:

F2b

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Dose descriptor:

LOAEL

Generation:

F2b

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

350 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Table 2: Overall results of the 2-generation study:

RELEVANT PARENTAL DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	50	160	500/350
Food Consumption
F0 males	-	-	-	-
F0 females	-	-	-	trans. reduced */**
F1 males	-	-	-	-
F1 females	-	-	-	-
Body Weight
F0 males	-	-	-	-
F0 females	-	-	-	trans. reduced */**
F1 males	-	-	-	-
F1 females	-	-	-	-
Relative Kidney Weight [%]
F0 males	6.58	6.66	6.82	7.45**
F0 females	7.68	7.56	7.67	7.98
F1 males	6.68	6.81	6.89	7.45**
F1 females	7.57	8.04	7.53	7.48
Relative Liver Weight [%]
F0 males	3.44	3.45	3.53	3.74**
F0 females	3.79	3.66	3.83	3.84
F1 males	3.53	3.46	3.49	3.70
F1 females	3.82	3.88	3.80	3.76
Relative Spleen Weight [%]
F0 males	1.44	1.67	1.47	1.44
F0 females	1.64	1.78	1.87*	1.94*
F1 males	1.42	1.41	1.44	1.56
F1 females	1.75	1.76	1.78	1.79
Histopathology
F0 males	-	-	-	-
F0 females	-	-	-	-
F1 males	-	-	-	-
F1 females	-	-	-	-
-: no effect, trans. reduced: transiently reduced, *: p<0.05, ** p<0.01

RELEVANT REPRODUCTIVE PERFORMANCE DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	50	160	500/350
Estrus to Estrus [days]
F0 females	2.8	2.7	2.8	2.5
F1 females	2.5	2.5	2.6	2.6
Mating Index [%]
F0 males/females (F1a)	93.3/93.3	100/100	93.3/93.3	100/100
F0 males/females (F1b)	93.1/93.3	93.1/93.1	86.2/86.7	93.3/93.3
F1 males/females (F2a)	100/100	96.6/96.7	96.7/96.7	96.7/96.7
F1 males/females (F2b)	96.7/96.7	96.6/93.1	93.3/93.3	93.3/93.3
Gestation Index [%]
F1a litter	100	96.2	100	100
F1b litter	100	100	100	100
F2a litter	100	100	100	100
F2b litter	100	100	100	100
Live Birth Index [%]
F1a litter	98.3	98.5	99.4	94.6*
F1b litter	98.3	98.5	98.9	98.9
F2a litter	99.3	99.5	99.3	99.2
F2b litter	96.4	97.7	98.9	100**
Litters with Liveborn but no Pups alive on Day 4 [%]
F1a litter	0	0	0	44.8
F1b litter	0	0	0	0
F2a litter	3.4	0	0	0
F2b litter	0	0	0	8.3
* p<0.05, ** p<0.01

RELEVANT DEVELOPMENTAL DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	50	160	500/350
Pups Delivered [mean]
F1a pups	13.9	13.1	14.2	12.7
F1b pups	14.8	14.1	15.7	14.0
F2a pups	13.9	15.0	14.3	13.1
F2b pups	15.6	15.2	14.6	13.8
Viability Index [%, day 0-4 p.p.]
F1a pups	95.3	97.6	98.8	43.3**
F1b pups	96.3	98.8	98.9	98.1
F2a pups	93.3	98.0**	97.8**	96.6
F2b pups	97.1	95.1	97.4	85.2**
Lactation Index [%, day 4-21 p.p.]
F1a pups	99.2	100	99.6	96.1
F1b pups	100	100	95.7**	98.5
F2a pups	98.5	99.3	99.6	98.8
F2b pups	99.2	98.8	98.2	92.0**
Pups Body Weight Gain
F1a pups	-	-	-	reduced **
F1b pups	-	-	-	-
F2a pups	-	-	-	init. reduced */**
F2b pups	-	-	-	reduced **
Sexual Maturation
Preputial Separation	-	-	-	-
Vaginal Opening	-	-	-	-
-: no effect, *: p<0.05, ** p<0.01, p.p.: post partum, init. reduced: initially reduced

The stability of NMP in the food over a period of up to 32 days at room temperature was proven. The correctness of the homogeneous distribution in the diet and the dietary concentrations were proven. The measured intakes of NMP by the different test groups correlated generally well with the desired target doses.

Conclusions:

In a two-generation reproduction toxicity study, NMP had no adverse effects on reproductive performance or fertility of the P0 or F1 parental animals of all substance-treated groups. All P0 and F1 parental rats proved to be fertile at least after one of the two mating intervals (P0 parents: F1a and F1b; F1 parents: F2a and F2b) as demonstrated by the clinical and histopathological examinations. There were no signs of systemic toxicity noted after high dose level reduction to 350 mg/kg bw/day. The F1a pups exposed to 500 mg/kg bw/day had a decrease in mean litter size, pup survival and pup body weights during lactation.The NOAEL (No-Observed-Adverse-Effect Level) for the parental and reproductive effects is 350 mg/kg bw/day. The NOAEL for the growth and development of the offspring is 160 mg/kg bw/day.

Executive summary:

In a two-generation reproduction toxicity study, groups of 30 Sprague-Dawley rats per sex were given NMP via the diet at initial dose levels of 0, 50, 160 or 500 mg/kg bw/day over a 10 -week premating period and throughout the mating, gestation, lactation and a rest period between pregnancies (Huntingdon Life Sci, 1999). The concentrations in the diet were adjusted regularly in respect to the actual body weight gain.

Due to severe pup mortality in the first litter (F1a), the highest dose level was reduced to 350 mg/kg bw/day for the further course of the study. Each generation gave birth to two litters. The parental animals for the second generation were selected from pups of the second litter (F1b).

NMP had no adverse effects on reproductive performance or fertility of the P0 or F1 parental animals of all substance-treated groups. All P0 and F1 parental rats proved to be fertile at least after one of the two mating intervals (P0 parents: F1a and F1b; F1 parents: F2a and F2b) as demonstrated by the clinical and histopathological examinations. There were no signs of systemic toxicity noted after high dose level reduction to 350 mg/kg bw/day. The F1a pups exposed to 500 mg/kg bw/day had a decrease in mean litter size, pup survival and pup body weights during lactation.

After reduction of the high dose level, F2b pups only at 350 mg/kg bw/day had a decrease in the number of pups surviving lactation and a decrease in pup body weights. At necropsy, parental animals revealed significant organ weight changes, however, they were considered not treatment-related due to the absence of changes in the other sex and the absence of corresponding histopathological findings.

Thus, the NOAEL for reproductive performance/fertility and systemic toxicity was 350 mg/kg bw/day. The NOAEL for developmental toxicity was 160 mg/kg bw/day.

This study is acceptable and satisfies the guideline requirement for a two -generation reproductive study in rats.

Reference 3

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

limited maternal information: body weight without information on body weight gain and food consumption; limited number of animals per group, protocol different from guidelines

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Source: E. I. du Pont De Nemours and Company, Richmond, Virginia, USA
- Purity: 99.9%
Impurities:
- 1.5 or isomer-dimethyl-2-pyrrolidone (986 ppm)
- N-methyl-succinimide (251 ppm)
- 2-pyrrolidinone (14 ppm)
- low boiling impurities (36 ppm), high boiling impurities (40 ppm).

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding North Wilmington, MA, USA
- Age at study initiation:
- first group (P0 generation): 21 days
- second group (unexposed mates for F1 generation): 64 days
- Weight at study initiation:
- first group: 32-50 g
- second group: 165-217 g
- Fasting period before study: not specified
- Housing:
- before exposure: individually, cages made of stainless steel wiremesh
- during exposure: individually, stainless steel cages placed within 750 or 900 liter glass and stainless steel chambers
- animal positions within the chambers were rotated daily
- Diet: ad libitum except during the inhalation exposures; Purina Certified Rodent Chow No. 5002 (Ralston Purina Company, St. Louis, MO, USA)
- Water: ad libitum, except during the inhalation exposures
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (72 ± 2°F)
- Humidity (%): 40-60
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: nitrogen carried vapors and was mixed with dilution air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: NMP pumped through inclined tube furnace heated to 195-210°C, N2 carried vapours throught the tube
- Method of holding animals in test chamber: hold in cages
- Source and rate of air: dilution air, 700 L/min
- Temperature, humidity, oxygen concentration: 18.9 - 27.7 °C (within control animal chambers: 19.2 - 28.3°C), about 50% r.h., below 21%
- Method of oxygen monitoring: Biomarine Oxygen Analyzer (Grade 1225R)
- Treatment of exhaust air: ventilation from bottom chamber through aqueous scrubbing tower

TEST ATMOSPHERE
- Brief description of analytical method used: HP 5710A GC with nitrogen/phosphorus detector
- Samples taken from breathing zone: no

Details on mating procedure:

P0
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: 5 days
- After 5 days of unsuccessful pairing replacement of first male by another male with proven fertilit of same treatment group.
- Further matings after two unsuccessful attempts: yes,if not mated female placed with another male of same treatment group for 5 d
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal lavage referred to as day 0 of pregnancy

F1 (on day 70 PP: postpartum)
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1 (by mating with additional control animals of the opposite sex)
- Length of cohabitation: 5 days
- After 5 days of unsuccessful pairing replacement of first male by another male with proven fertilit of same treatment group.
- Further matings after two unsuccessful attempts: yes,if not mated female placed with another male of same treatment group for 5 d
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal lavage referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test atmosphere measured with HP 5710A GC with nitrogen/phosphorus detector.

Duration of treatment / exposure:

Exposure period: males: 100 d, females: 147 - 177 d
Premating exposure period (males): 85 d
Premating exposure period (females): 85 d
Duration of test: approx. 177 d

Frequency of treatment:

6 h/d, 7x/wk

Details on study schedule:

- F1 parental animals not mated until Day 77 PP after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 35 days of age.

Dose / conc.:

10.3 ppm (analytical)

Remarks:

equivalent to 41 mg/m3

Dose / conc.:

50.8 ppm (analytical)

Remarks:

equivalent to 206 mg/m3

Dose / conc.:

116.4 ppm (analytical)

Remarks:

equivalent to 478 mg/m3

No. of animals per sex per dose:

groups of 10 males and 20 females

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- P0 upon arrival and twice more before exposure
- during exposure while in the chamber and during handling

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
- druing handling (remove from chamber and return into chamber)
- P0 males: weekly for recovery animals
- mated females: Day 1-21 gestation
- dams: pathological changes
- F1 rats on Days 1, 4, 14, and 21 PP: postpartum, weekly for males until euthanization; females until mating
- Mated F1: Days 1-21 GD: gestation day and 1 and 22 PP

BODY WEIGHT: Yes
- Time schedule for examinations:
- P0 upon arrival and twice more before exposure
- P0 males weekly until euthanization; females weekly until mating and on d1 and d21 of gestation
- F1 males: Days 1, 4, 14, and 22 PP, weekly until they were euthanized
- F1 females: Days 1, 4, 14, 21 PP, weekly until mating and on Day 1 and 21 GD and on Days 1 and 22 PP
- F2: Days 1 and 2 PP

Sperm parameters (parental animals):

Parameters examined in P/F1 male parental generations:
testis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, 4/sex/litter as nearly as possible; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, livebirths, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after breeding for the P0 generation and on Day 85 PP for the F1 generaion.
- Female animals: All surviving animals on Day 21 PP for P0 generation and on Day 2 PP for the F1 generation.

GROSS NECROPSY
- Gross changes of reproductive organs observed for F1 generation.
- Gross pathological changes observed for the pups of the P0 generation within the developmental study part (Solomon et al., 1995).

POST-MORTEM EXAMINATIONS: Yes
- Organs examined: ovarian weight

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 and 2 days of age, respectively.
- These animals were subjected to postmortem examinations as follows:

GROSS NECROPSY
- Gross necropsy consisted of reproductive organs.

Statistics:

Fisher's exact test with Cochran-Armitage (pregnancy rate, clinical signs, maternal deaths, mating, fertility, gestation indices, litter survival) 
ANOVA and Dunnett's test (gestation length, body weight data, liver weights)
Mann-Whitney U test (nidations)

Reproductive indices:

Female / Male Mating Index (%) = (Number copulating / Number cohoused) * 100
Female[a] / Male[b] Fertility Index (%) = (Number bearing[a] litters sired[b] / Number copulating) * 100
Gestation index (%) = (Number of litters with at least on life pup / Number of litters) * 100

Offspring viability indices:

Viability Index (%) = (Number of pups alive Dav 4 Pre-standardization/pooling / Dav 4 Pre-standardization/pooling) * 100
Lactation Index (%) = (Number of pups alive at Weaning [Day 21 postpartum] / Number of pups alive Day 4 after litter standardization/pooling) * 100

Clinical signs:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- P0 rats died during the study: two in the control group and one in the 51 ppm group
- 1/2 control rats died from injuries during handling

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Food efficiency:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- exposure-related reduction in response to sound was noted at 116 ppm

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

No effects on reproductive ability/organs were observed.
Besides, in the devlopmental part of the study, no effects apart from slightly reduced pup body weight were observed.

Dose descriptor:

NOAEC

Effect level:

51 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Behaviour

Remarks on result:

other: equivalent to 206 mg/m3

Dose descriptor:

LOAEC

Effect level:

116 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Behaviour: exposure-related reduction in response to sound was noted

Remarks on result:

other: equivalent to 478 mg/m3

Clinical signs:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- body weight gain of the F1 offspring was reduced when both parents inhaled 116 ppm

Food efficiency:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

51 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: equivalent to 206 mg/m3

Dose descriptor:

LOAEC

Generation:

F1

Effect level:

116 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: equivalent to 478 mg/m3

Critical effects observed:

no

Reproductive effects observed:

no

Lowest effective dose / conc.:

51 ppm

Treatment related:

no

Table 1: Delivery and litter data of the P0 and P1 generations (ECHA, Background document to the Opinion on NMP, 2014).

 

	Dose (mg / m^3)
	0	41	206	478	478 (only females exposed)	478 (only males exposed)
PO generation
Natural delivery and litter data
Male mating index (%)	27/30 (90)	8/10 (80)	9/10 (90)	18/20 (90)	10/10 (100)	9/10 (90)
Male fertility index (%)	27/27 (100)	8/8 (100)	8/9 (88,9)	17/18 (94,4)	10/10 (100)	8/9 (88,9)
Female mating index (%)	56/58 (96,6)	18/20 (90)	18/19 (94,7)	40/40 (100)	20/20 (100)	20/20 (100)
Female fertility index (%)	53/56 (94,6)	16/18 (88,9)	15/18 (83,3)	37/40 (92,5)	15/20 (75)	17/20 (85)
Gestation index (%)	53/53 (100)	16/16 (100)	15/15 (100)	37/37 (100)	15/15 (100)	17/17 (100)
Mean gestation length N	34	14	14	19	13	15
X (days)	22,6	22,5	22,6	22,7	22,7	22,6
SE	0,08	0,14	0,14	0,11	0,13	0,12
Mean number of offspring/litter
-Born	13,7	14,0	13,7	14,2	13,9	14,7
-Born alive	13,5	13,9	13,5	14,1	13,9	14,6
-Day 4 PP preculling	13,4	13,9	13,3	13,9	13,6	14,3
-Day 4 PP postculling	8,0	7,9	7,9	8,0	8,0	7,9
-Day 14 PP	8,0	7,9	7,9	8,0	8,0	7,9
-Day 21 PP	8,0	7,8	7,9	8,0	8,0	7,9
Viability index (%)	99,3	100,0	98,6	98,3	98,0	97,7
Lactation index (%)	100,0	98,4	99,0	100,0	100,0	100,0
Sex ratio (% males)	0,53	0,46*	0,43	0,46	0,48	0,50
Offspring weight/litter (g)
-Day 1 PP	7,5	7,0*	7,1	6,7*	7,1	7,3
-Day 4 PP preculling	10,8	10,0*	10,3	9,6*	10,1	10,5
-Day 4 PP postculling	10,7	9,9*	10,2	9,6*	9,9*	10,6
-Day 14 PP	30,8	27,8*	29,5	28,7*	28,6*	32,0
-Day 21 PP	49,1	45,6*	47,4	46,9*	47,2	51,6*
F1 generation
Male mating index (%)	20/20 (100)	15/16 (93.8)	15/15 (100)	22/22 (100)
Male fertility index (%)	18/20 (90)	14/15 (93,3)	14/15 (93,3)	19/22 (86,4)
Female mating index (%)	19/20 (95)	15/16 (93,8)	15/15 (100)	22/22 (100)
Female fertility index (%)	18/19 (94,7)	14/15 (93,3)	14/15 (93,3)	19/22 (86,4)
	Dose (mg / m^3)
	0	41	206	478	478 (only females exposed)	478 (only males exposed)
Gestation index (%)	17/18 (94,4)	14/14 (100)	14/14 (100)	19/19 (100)
Data from exposed females mated to unexposed males
Mean number of offspring/litter
-Born	14,7	16,1	15,7	13,9
-Born alive	14,7	15,9	15,7	13,8
-Day 2 PP	14,6	15,9	15,6	13,8
Offspring weight/litter (g)
-Day 1 PP	6,7	6,5	6,7	6,9
-Day 2 PP	7,5	7,3	7,5	7,7
Mean number of offspring/litter
-Born	13,2	13,6	14,0	13,7
-Born alive	13,2	13,5	14,0	13,6
-Day 2 PP	13,2	13,3	14,0	13,6
Offspring weight/litter (g)
-Day 1 PP	6,4	6,3	6,4	6,5
-Day 2 PP	7,3	7,2	7,2	7,3

 * significantly different from control, p ≤ 0.05

Table 2: Final body weights, testes and ovarian weights (ECHA, Background document to the Opinion on NMP, 2014).

 

Concentration (mg / m^3)	N	Final body weight (g)	Testes weight (g)	Relative testes weight
Mean testes weight
PO generation
0	20	529 (10,5)	3,47 (0,76)	0,66 (0,016)
41	5	547 (9,8)	3,56 (0,251)	0,65 (0,041)
206	5	559 (25,4)	3,65 (0,113)	0,66 (0,018)
478	15	530 (6,7)	3,60 (0,074)	0,68 (0,017)
F1 generation
0	20	439 (11,4)	3,36 (0,094)	0,77 (0,013)
41	16	448 (5,8)	3,62 (0,139)	0,81 (0,030)
206	15	457 (11,4)	3,35 (0,082)	0,74 (0,019)
478	22	449 (7,8)	3,42 (0,049)	0,77 (0,015)
Mean ovarian weight
PO generation
0	56	369,8 (3,41)	142 (4,08)	38 (1,06)
41	16	361,8 (4,77)	131 (6,98)	36 (1,79)
206	15	360,9 (7,15)	133 (7,15)	37 (1,79)
478*	36	369,4 (4,16)	136 (6,08)	37 (1,71)
Concentration (mg / m3)	N	Final body weight (g)	Testes weight (g)	Relative testes weight
F1 generation
0	17	315,8 (4,65)	189 (6,39)	60 (2,57)
41	14	320,5 (7,40)	218 (9,79)	69 (2,75)
206	14	330,2 (8,81)	210 (8,58)	64 (2,25)
478	19	318,9 (6,27)	198 (6,55)	62 (2,33)

 

Conclusions:

The toxicological significance of the apparent reduced responsiveness of top dose animals to sound is not established, but at most it represented a slight narcotic effect, the parental NOAEC therefore appears to be 50 ppm vapor. In the offspring, there was a slight decrease in pup body weight at the highest concentration only.

Overall, the study provides no evidence of an adverse effect on any parameters of fertility.

Thus, it is assessed that the following NOAECs were achieved:

Reproductive performance and fertility:
116 ppm (LOAEC), equal to 478 mg/m3
Parental toxicity:
50 ppm, equal to 206 mg/m3

Executive summary:

In a two-generation reproduction study in rats 10 males and 20 females per dose level were exposed whole body to 0, 10 ppm (41 mg/m3), 51 ppm (206 mg/m3) or 116 ppm (478 mg/m3) of NMP vapour (Solomon et al., 1995). Sprague-Dawley rats were exposed for 6h/day, 7 days/week, for a minimum of 14 weeks. Two satellite groups, where either male or female animals were exposed, were tested additionally at 478 mg/m3.

Besides, a developmental study was conducted which is described in chapter Developmental toxicity / teratogenicity.

Animals were mated after 12 weeks of exposure to NMP vapours and both parents and offspring were examined for effects on reproductive organs /reproduction. The F1 generation was not exposed after weaning and the F2 generation was produced by mating with additional rats (non-experimental animals) of the opposite sex.

There were no effects observed for the reproducibility or the reproductive organs. For the developmental effects, apart from reduced body weight of the pups at the high dose, no adverse effects were observed (Solomon et al., 1995). This observation was in line with reduced body weight gain of the F1 generation whose parents were exposed to 478 mg/m3. The effects persisted till 21 days after birth. There is no clear dose response for this effect discussed because the effects were missing for the 206 mg/m3 dose group.

For maternal toxicity a NOAEC of 51 ppm (206 mg/m3) is proposed. However, as the only effect in maternal animals was the reduced sensitivity to sound in parental animals before mating, this effect is considered to be a systemic toxicity effect.

The developmental LOAEC is 116 ppm (478 mg/m3) as effects to the pup body weight in the developmental part of the study (Solomon et al., 1995) and reduced body weight gain for the F1 generation is described in the study.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

478 mg/m³

Study duration:

chronic

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Oral route:

In a two-generation reproduction toxicity study (BASF SE, 1999), groups of 25 Wistar rats per sex were given NMP via the diet at initial dose levels of 0, 50, 160 or 500 mg/kg bw/day over a 10 -week premating period and throughout the mating, gestation, lactation and a rest period between pregnancies. The concentrations in the diet were adjusted regularly in respect to the actual body weight gain. Due to severe pup mortality in the first litter (F1a), the highest dose level was reduced to 350 mg/kg bw/day for the further course of the study. Each generation gave birth to two litters. The parental animals for the second generation were selected from pups of the second litter (F1b). NMP had no adverse effects on reproductive performance or fertility of the F0 or F1 parental animals of all substance-treated groups. All F0 and F1 parental ratsproved to be fertile at least after one of the two mating intervals (F0 parents: F1a and F1b; F1 parents: F2a and F2b) as demonstrated by the clinical and histopathological examinations. There were signs of systemic toxicity in each of the high dose groups at 500 mg/kg bw/day and also after reduction to 350 mg/kg bw/day. Parental toxicity consisted of reduced body weight gain and food intake as well as kidney findings in form of impaired organ weight and histopathological findings. Developmental toxicity was evidenced by increased pup mortality and reduced body weight gain, including corresponding effects in the investigated organs, in pups treated at 500/350 mg/kg bw/day. Thus, the NOAEL for reproductive performance/fertility was 350 mg/kg bw/day. The NOAEL for systemic (parental) and developmental toxicity was 160 mg/kg bw/day.

In a second two-generation reproduction toxicity study (Huntingdon Life Science, 1999), groups of 30 Sprague-Dawley rats per sex were given NMP via the diet at initial dose levels of 0, 50, 160 or 500 mg/kg bw/day over a 10 -week premating period and throughout the mating, gestation, lactation and a rest period between pregnancies. The concentrations in the diet were adjusted regularly in respect to the actual body weight gain. Due to severe pup mortality in the first litter (F1a), the highest dose level was reduced to 350 mg/kg bw/day for the further course of the study. Each generation gave birth to two litters. The parental animals for the second generation were selected from pups of the second litter (F1b). NMP had no adverse effects on reproductive performance or fertility of the F0 or F1 parental animals of all substance-treated groups. All F0 and F1 parental rats proved to be fertile at least after one of the two mating intervals (F0 parents: F1a and F1b; F1 parents: F2a and F2b) as demonstrated by the clinical and histopathological examinations. There were no signs of systemic toxicity noted after high dose level reduction to 350 mg/kg bw/day. The F1a pups exposed to 500 mg/kg bw/day had a decrease in mean litter size, pup survival and pup body weights during lactation. After reduction of the high dose level, F2b pups only at 350 mg/kg bw/day had a decrease in the number of pups surviving lactation and a decrease in pup body weights. At necropsy, parental animals revealed significant organ weight changes, however, they were considered not treatment-related due to the absence of changes in the other sex and the absence of corresponding histopathological findings. Thus, the NOAEL for reproductive performance/fertility and systemic toxicity was 350 mg/kg bw/day. The NOAEL for developmental toxicity was 160 mg/kg bw/day.

The findings of the above 2-generation studies are further supported by publications of Sitarek et al. from 2008 and 2012. The influence of NMP on both the male and the female fertility was examined as well as the changes in development of progeny after oral gavage of NMP at concentrations of 100, 300, 1000 mg/kg bw/d (males) and 150, 450 and 1000 mg/kg bw/d (females). After mating of treated animals with untreated animals of the opposite sex, the fertility index, viability index and lactation index were determined (Sitarek et al, 2008, 2012).

Inhalative route:

In a two-generation reproduction study in rats 10 males and 20 females per dose level were exposed whole body to 0, 10 ppm (41 mg/m3), 51 ppm (206 mg/m3) or 116 ppm (478 mg/m3) of NMP vapour. Sprague-Dawley rats were exposed for 6h/day, 7 days/week, for a minimum of 14 weeks. Two satellite groups, where either male or female animals were exposed, were tested additionally at 478 mg/m3.

Besides, a developmental study was conducted (Solomon et al., Drug. Chem. Toxicol. 18, 1995) which is described in chapter Developmental toxicity / teratogenicity.

Animals were mated after 12 weeks of exposure to NMP vapours and both parents and offspring were examined for effects on reproductive organs /reproduction. The F1 generation was not exposed after weaning and the F2 generation was produced by mating with additional rats (non-experimental animals) of the opposite sex.

There were no effects observed for the reproducibility or the reproductive organs. For the developmental effects, apart from reduced body weight of the pups at the high dose, no adverse effects were observed (Solomon et al., Drug. Chem. Toxicol. 18, 1995). This observation was in line with reduced body weight gain of the F1 generation whose parents were exposed to 478 mg/m3. The effects persisted till 21 days after birth. There is no clear dose response for this effect discussed because the effects were missing for the 206 mg/m3 dose group.

For maternal toxicity a NOAEC of 51 ppm (206 mg/m3) is proposed. However, as the only effect in maternal animals was the reduced sensitivity to sound in parental animals before mating, this effect is considered to be a systemic toxicity effect.

The developmental LOAEC is 116 ppm (478 mg/m3) as effects to the pup body weight in the developmental part of the study (Solomon et al., Drug. Chem. Toxicol. 18, 1995) and reduced body weight gain for the F1 generation is described in the study.

Effects on developmental toxicity

Description of key information

Two oral, four inhalative and two dermal prenatal developmental toxicity studies in line with the requirements of OECD 414 are used to assess the developmental/teratogenicity hazard of NMP.

Oral route:

The oral studies were performed in Sprague-Dawley rats and in New Zealand white rabbits and exposed by oral gavage.

The oral study was performed in Sprague-Dawley rats, treated with 125, 250, 500 or 750 mg/kg bw/day during gestational day 6 through 20. Significant decrements in maternal body weight gain and food consumption occurred at 500 mg/kg bw/day. The developmental toxicity was seen for increased implantation losses and number of resorptions at 500 mg/kg bw/day. At this concentration, the fetal rate of malformations was increased and a delayed ossification of skull bones and sternebrae was observed. The fetal weights were reduced at 250 mg/kg bw/day onwards. The NOAEL for maternal toxicity and developmental toxicity was 125 mg/kg bw/day, whereas a NOAEL of 250 mg/kg bw/day for malformations was defined (Saillenfait et al., 2002).

In a second study performed at the laboratories of the IRDC, 20 inseminated rabbits were administered 55, 175 or 540 mg/kg bw/day NMP daily on gestation day 6 through 18. Significantly reduced body weight and reduced food consumption was observed at 175 and 540 mg/kg bw/day in maternal animals. Developmental effects, e.g. increased implantation loss, increased implantation resorptions and a higher incidence of malformations were observed in the high dose group. At the lower dose level, no embryo-/fetotoxic effects or malformations were noted. The NOAEL for maternal as well as developmental toxicity was 175 mg/kg bw/day (IRDC, 1991).

Inhalative route:

The inhalation studies were performed in Sprague-Dawley rats and Himalayan rabbits exposed in whole body. Two supporting studies used Wistar rats which are discussed under ‘Additional Informations’.

Lee et al. observed neither developmental nor maternal toxicity in pregnant Sprague-Dawley rats exposed 6h/d from gestation day 6 through 15. The overall NOAEC was 0.36 mg/L (87 ppm) (Lee et al., 1987).

In another study with Sprague-Dawley rats, Saillenfait et al. observed decreased maternal body weight and food consumption at 60 and 120 ppm as well as decreased fetal body weights for dams exposed to 120 ppm. The dams were exposed 6h daily from gestational day 6 through 20. The NOAEC for maternal effects was reported to be 30 ppm and for developmental effects to be 60 ppm (Saillenfait et al., 2003).

Solomon et al. exposed female Sprague-Dawley rats until 20 days of gestation as part of a reproduction toxicity study (Solomon et al., 1995) daily with NMP. A decreased response to sound was the only observed effect on maternal toxicity and only a slight decrease in pup weight was observed at 116 ppm. Based on the reported NOAEC in the reproduction toxicity study of 206 mg/m3 (51 ppm) and the limited effect of reduced sensitivity to sound, this NOAEC is considered the systemic NOAEC. The LOAEC for developmental toxicity was 116 ppm but a NOAEC of 206 mg/m3 (51 ppm) is also described in the reproductive toxicity study based on decreased pup weight in the F1 offspring.

A study in Himalayan rabbits that were exposed to 0.2, 0.5 and 1.0 mg/L (49, 122, 143 ppm) was performed by BASF. The inseminated animals were exposed for 6 hours daily on gestation day 7 through 19 (BASF SE, 1993). No maternal toxicity is described in the study at any concentration, however in the range-finding study (BASF SE, 1991) increased liver weights and impaired clinical parameters are discussed at 1.0 and 2.0 mg/L. The increased number of fetuses with accessory 13th rib(s) in the high dose group was assessed to be a non-specific effect. Therefore, the NOAEC for maternal and developmental toxicity was 0.5 mg/L (122 ppm).

The most sensitive adverse effect from all studies via the inhalation route is a reduction in fetal/pup body weight which was also agreed by an opinion paper by RAC and SCOEL (RAC and SCOEL, Joint Opinion for N-Methyl-2-Pyrrolidone, 30 November 2016).

The NOAEC in the study from Lee et al. (1987) is used as point of departure (POD) for the later DNEL calculation as it is below the lowest LOAEC of the Solomon study of 116 ppm (478 mg/m3) and the below the LOAEC (618 mg/m3 ~ 150 ppm) of a supporting study of Hass et al. (1995). The reported findings in two studies by Hass et al. were delayed physical development and 6-7% reduction in fetal body weight.

Dermal route:

The two studies were performed in Sprague-Dawley rats and Himalayan rabbits.

A study by the GAF Corporation investigated NMP after dermal application of 75, 237 or 750 mg/kg bw/day in 25 Sprague Dawley rats. The animals were exposed under open conditions for eight hours daily from gestation day 6 through 15. Reduced maternal body weight was observed at 750 mg/kg bw/day together with topical irritations. Fewer live fetuses, increased resorption rates, reduced fetal weight and retarded skeletal development were observed as markers of fetal toxicity at the high dose. A NOAEL of 237 mg/kg bw/day was described for maternal and developmental toxicity as well as teratogenicity (GAF Corp., 1979).

A study in Himalayan rabbits that were dermally exposed to a 40% aq. solution at 100, 300 or 1000 mg/kg bw /day was performed. No maternal toxicity was observed and no developmental toxicity apart from a slight increases of a variation of the 13th rib(s) at 1000 mg/kg bw/day. The NOAEL for maternal toxicity and teratogenicity was 1000 mg/kg bw/day. The NOAEL for developmental toxicity was 300 mg/kg bw/day (BASF SE, 1993).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-01-24 to 1991-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Atrix Laboratories, Fort Collins, Colorado, USA and 9351-116
- Expiration date of the lot/batch: 18 February 1991
- Analytical purity: 99 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, light occlusive container under a blanket of dry nitrogen
- Stability under test conditions: at room temperature: 18 February 1991

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: preparing a solution with deionized water by using a magnetic stir plate

FORM AS APPLIED IN THE TEST: diluted solution

Species:

rabbit

Strain:

New Zealand White

Remarks:

SPF rabbits

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton-LRE, Kalamazoo, Michigan, USA
- Age at study initiation: 26 - 27 weeks old, at insemination
- virgin females
- Weight at study initiation: 3561 - 4588 g on gestation day 0
- Housing: singly, suspended stainless steel cages with deotized animal cage boards
- Diet: commercial diet (certified rabbit chow #5322, Purina Mills, St Louis, Missouri, USA), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 57 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 (62 - 73 °F)
- Humidity (%): 49 - 72
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours

IN-LIFE DATES: From: 10 December 1990 To: 08 March 1991

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

intragastric intubation

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test material was weighed and dissolved in the vehicle, deionized water, using a magnetic stir bar and stir plate. The solution was transferred to a volumetric flask, and additional vehicle was added to yield the appropriate amount of prepared test article. The flask was shaken manually, and the contents dispensed into amber glass containers and stored at room temperature.
The test article was prepared weekly at concentrations to permit the administration of dose levels of 55, 175 and 540 mg/kg bw/day at a dose volume of 3.0 mL/kg .

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing preparations which were stored at room temperature for 10 days were analyzed to verify the stability of the dosing solutions for at least one week.
Dosing solutions were analyzed twice during the study to ensure the correct concentrations of the test article. The first analysis was conducted early during the dosing period, and the second analysis was conducted at approximately the middle of the dosing period.

Details on mating procedure:

- Impregnation procedure: artificial insemination
Nine proven male stock rabbits of the same breed and source were selected to serve as sperm donors.
Semen was collected using an artificial vagina; the gelatinous plug was removed from the ejaculate. The semen was evaluated for motility and was used for insemination only if the motility was 60% or greater as determined by inspection.
Within one hour after insemination, ovulation was induced by an injection of 100 U.S.P. units of human chorionic gonadotropin into a marginal ear vein.
Semen from one male was used to inseminate two or three females in each group each day. The day of insemination was designated as gestation day 0.

Duration of treatment / exposure:

day 6 through 18 of gestation

Frequency of treatment:

1x/day

Duration of test:

29 days

Dose / conc.:

55 mg/kg bw/day (actual dose received)

Dose / conc.:

175 mg/kg bw/day (actual dose received)

Dose / conc.:

540 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a range-finding developmental toxicity study in New Zealand White Rabbits the dosage levels were selected

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily on GD 6-29, 2x/d for mortality and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on gestation days 0, 6, 12, 19, 24 and 29.

FOOD CONSUMPTION: Yes
- Time schedule: measured daily for each individual and summarized for the intervals of gestation days 0-6, 6-12, 12-19, 19-24, 24-29, 6-19 and 0-29.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: ovaries and uterus, gross necropsy

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of dead fetuses

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No data

Statistics:

ANOVA and Dunnett's test
Fisher's exact test
Kruskal-Wallis test

Indices:

None

Historical control data:

Historical control data were included in the report.

Clinical signs:

not specified

Description (incidence and severity):

- no overt clinical signs of toxicity at any dose

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

175: body weight gain was slightly but significantly reduced only for gestation days 6-12
540 mg/kg bw/day: significantly reduced body weight gain at both doses (marked at 540 mg/kg bw/day)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

540 mg/kg bw/day: reduced feed consumption

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

540 mg/kg bw/day: reduced mean uterine weight

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

540 mg/kg bw/day: one abortion

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

540 mg/kg bw/day: increased post-implantation loss, due to increased early and late resorptions

Total litter losses by resorption:

not examined

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

540 mg/kg bw/day: increased early and late resorptions

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

540 mg/kg bw/day: reduced live litter size

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Dose descriptor:

NOAEL

Effect level:

55 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: The effects were observed at higher concentrations and appear to follow a dose response relationship.

Dose descriptor:

LOAEL

Effect level:

175 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Abnormalities:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

55 and 175 mg/kg bw/day: incidences and types of fetal malformations were comparable to those in the control group

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

55 and 175 mg/kg bw/day: incidences and types of fetal malformations were comparable to those in the control group
540 mg/kg bw/day: malformed skull bones

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

55 and 175 mg/kg bw/day: incidences and types of fetal malformations were comparable to those in the control group
540 mg/kg bw/day: increases in cardiovascular malformations: bulbous aortic arch, pulmonary trunk stenosis, ductus arteriosus stenosis, and interventricular septal defect

Dose descriptor:

NOAEL

Effect level:

175 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

Dose descriptor:

LOAEL

Effect level:

540 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

visceral/soft tissue: cardiovascular

Description (incidence and severity):

only in the high-dose group increased in comparison with the high-dose group

Developmental effects observed:

yes

Lowest effective dose / conc.:

540 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

no

Relevant for humans:

not specified

Test substance analysis: Stability of the solutions was demonstrated since the mixtures stored at room temperature for 10 days contained 96 - 100 % of the initial concentrations. The correctness of the concentrations was demonstrated by recovery values between 96 - 102 %.

Table 1: Maternal and gestational gestational parameters from pregnant New Zealand White rabbits as well as incidence of malformations in foetuses.

MATERNAL DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	55	175	540
Inseminated females [n]	20	20	20	20
Pregnant females [n]	20	18	18	17
Mortality of does [n]	0	0	0	0
Food consumption (days 6-19) [g]	173	164	165	144*
Body weight change (days 6-19) [g]	151	165	87	6*
*: p < 0.05

CAESARIAN SECTION / FETAL EXAMINATION
Endpoint	Dose Group [mg/kg bw/day]
	0	55	175	540
Inseminated females [n]	20	20	20	20
Pregnant dams [n]	20	18	18	17
Aborted [n]	0	0	0	1
Dams with viable fetuses [n]	20	17	18	15
Dams with all resoprtions [n]	0	1	0	1
Uterus weight, mean [g]	470.7	460.7	402.3	356.7
Corpora lutea, mean [n]	13.6	14.1	13.5	13.5
Implantation sites, mean [n]	9.1	7.6	6.7	7.0
Live fetuses / litter, mean [n]	8.6	6.8	6.0	5.2
Total resorptions / litter, mean	0.3	0.5	0.7	1.8
Early resorptions / litter, mean	0.3	0.4	0.5	1.4*
Late resorptions / litter, mean	0	0.1	0.2	0.4*
Dead fetuses [n]	1	6	0	0
Fetal weights, males, mean [g]	42.0	46.8	47.5	43.3
Fetal weights, females, mean [g]	41.4	43.9	45.3	41.0
Fetuses (litter) with malformations [n]	9 (7)	5 (5)	10 (7)	36 (12)*
Fetuses (litter)with variations [n]	126 (19)	101 (17)	102 (17)	82 (15)
*: p < 0.05

Conclusions:

The no observable adverse effect level (NOAEL) of NMP when administered orally to inseminated New Zealand White Rabbits was equivocally considered to be 55 mg/kg/day with respect to maternal toxicity, and 175 mg/kg/day with respect to developmental toxicity.
As the maternal effects were observed at higher concentrations and appear to follow a dose response relationship, the effects seen at 175 mg/kg bw/d may also be taken into account in setting the NOAEL for maternal toxicity.

Executive summary:

In this developmental toxicity study, groups of 20 inseminated New Zealand White rabbits were administered by oral gavage dose levels of 0, 55, 175 and 540 mg/kg bw/day of an aqueous NMP solution on gestation day 6 through 18 (IRDC, 1991).

Maternal toxicity was present at 175 and 540 mg/kg bw/day, expressed as significantly reduced body weight gain at both doses (marked at 540 mg/kg bw/day), reduced feed consumption at 540 mg/kg bw/day, and one abortion at 540 mg/kg bw/day. At 175 mg/kg bw/day, maternal body weight gain was transiently reduced on gestation days 6-12.

Developmental toxicity was observed at 540 mg/kg bw/day in form of increased post-implantation loss, due to increased early and late resorptions, reduced live litter size, and reduced mean uterine weight.

Malformations observed at 540 mg/kg bw/day were related to the cardiovascular system and the skeleton (skull bones). No embryo-/fetotoxic effects or malformations were noted at lower dose levels.

NOAEL for maternal toxicity is 55 mg/kg bw/day. As the maternal effects were observed at higher concentrations and appear to follow a dose response relationship, the effects seen at 175 mg/kg bw/d may also be taken into account in setting the NOAEL for maternal toxicity. The NOAEL of 175 mg/kg bw/day was assumed for both developmental toxicity and malformations.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

not specified

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: > 99.5 %
- Source: Merck, Darmstadt, Germany

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories, Saint-Germain-sur-l'Arbresle, France
- Age at study initiation: young adult
- Weight at study initiation: 200 - 220 g (females)
- Housing: after impregnation: single, clear polycarbonate cages with stainless-steel wire lids and corn cob granules as bedding
- Diet: commercial diet (pellets, UAR Alimentation, Villemoisson, France), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 1 - 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Humidity (%): 50 ± 5
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled water, gastric intubation

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were formulated in distilled water. The dosing volume was 5 mL/kg.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: yes, adult males from the same strain and supplier
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation

Duration of treatment / exposure:

day 6 through day 20 of gestation

Frequency of treatment:

1x/day

Duration of test:

21 days

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

750 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25-27 (21-25 pregnant)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of two exploratory dose range-finding studies using doses of 500, 1000 and 1500 mg/kg bw or 500, 625 and 750 mg/kg bw.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0, 6, 9, 12, 15, 18 and 21

FOOD CONSUMPTION: Yes
- Time schedule: measured at 3-day intervals starting on gestation day 6

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: ovaries and uterus, gross necropsy

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Other: dead and live fetuses

Fetal examinations:

- External examinations: Yes: all per litter, including the oral cavity
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

ANOVA and Dunnett's test: corpora lutea, implantation sites, live fetuses, body weight
Kruskal-Wallis test followed by Mann-Whitney test: post-implantation loss, dead fetuses, resorption, alterations among litters
Fisher's test: pregnancy rate, incidences of fetal alterations per dose

The litter was used as the basis for the analysis of fetal variables. The reported level of statistical significance was P<0.05.

Indices:

None

Historical control data:

None

Clinical signs:

no effects observed

Description (incidence and severity):

There were no effects observed compared to the control group.
Discoloration of urine was observed among all treated dams as indication of systemic availability of NMP.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

500 and 750 mg/kg bw/day: statistically significant reductions in maternal body weight gain occurred during the dosing period (gestation days 6-21), the maternal weight was reduced by 9% at 250 mg/kg bw/day, in addition the net weight change in dams was reduced by 11%, 26% and 26% at 250, 500 and 750 mg/kg bw/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

500 and 750 mg/kg bw/day: statistically significant reductions in food consumption occurred during the dosing period (gestation days 6-21)

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related effects were noted on number of corpora lutea and implantation sites.
500 mg/kg bw/day onwards: post-implantation losses and incidence of resorptions were increased with a steep dose-response relationship.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day onwards: post-implantation losses and incidence of resorptions were increased with a steep dose-response relationship.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw/day: only 8/25 dams had live fetuses

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No treatment-related effects were noted on pregnancy rate

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (actual dose received)

Basis for effect level:

body weight and weight gain

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

250 mg/kg bw/day onwards: significant reductions in fetal body weight for all sexes were observed

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw/day: number of live fetuses was markedly reduced

Changes in sex ratio:

no effects observed

External malformations:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day onwards: statistically significant increase in the incidences of external malformations

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day onwards: increase in the incidences of skeletal malformations

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw/day: statistically significant increase in the incidences of visceral malformations

Other effects:

effects observed, treatment-related

Description (incidence and severity):

250 mg/kg bw/day onwards: retarded development as indicated by body weight reduction was also accompanied with reduced ossification of the skull and sternebrae.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

effects observed, treatment-related

Localisation:

external: anus

skeletal: vertebra

visceral/soft tissue: cardiovascular

other: external: anasarca

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Maternal and gestational gestational parameters from pregnant Sprague-Dawley rats as well as incidence of malformations in foetuses of Sprague-Dawley rats given NMP.

MATERNAL DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	125	250	500	750
Mated females [n]	27	27	25	26	26
Pregnant females [n]	21	22	24	25	25
Mortality of dams [n]	0	0	0	0	0
Clinical symptoms	-	discol. urine	discol. urine	discol. urine	discol. urine
Food consumption (days 6-21) [g]	26	26	25	24**	22**
Body weight gain (days 6-21) [g]	134	134	122^#	102**	65**
Net body weight change [g]	38	40	34^#	28*	28*
-: no effect, disc.: discolored, *: p < 0.05, **: p < 0.01 ^#: although not statistically significant, biological relevance should be considered as the 9 % reduction is in line with statistically significantly reduced fetal weights at the same extend.

CAESARIAN SECTION / FETAL EXAMINATION
Endpoint	Dose Group [mg/kg bw/day]
	0	125	250	500	750
Females mated [n]	27	27	25	26	26
Pregnant dams [n]	21	22	24	25	25
Dams with viable fetuses [n]	21	21	24	25	8
Corpora lutea, mean [n]	14.6	14.6	14.3	14.5	14.8
Implantation sites, mean [n]	13.3	13.6	13.3	14.0	13.8
Post implantation loss, mean [%]	4.1	9.3	4.5	10.6*	94.2**
Dead fetuses / litter, mean [%]	0	0.4	0	1.2	3.2
Resorption sites / litter, mean [%]	4.1	8.9	4.5	9.4*	91.0**
Live fetuses / litter, mean [n]	12.7	13.1	12.7	12.4	2.4**
Fetal weights, males, mean [g]	5.79	5.74	5.32**	4.18**	3.03**
Fetal weights, females, mean [g]	5.62	5.47	5.02**	3.88**	3.09**
External malformations [%]	0	0.4	0.3	3.5**	15.8**
Visceral malformations [%]	0.7	0	0	6.4*	60**
Skeletal malformations [%]	0	0	0	9.0**	55.6**
External variations [%]	0	0.4	0.3	0.6	0
Visceral variations [%]	3.0	0	1.3	3.2	30.0**
Skeletal variations [%]	24.8	23.9	27.0	74.2**	100.0**
*: p < 0.05, **: p < 0.01

Conclusions:

Post implantation losses and the number of resorptions were increased at 500 mg/kg bw/day, showing a steep dose-response relationship. At the same concentration and onwards, the rate of fetal malformations (external, skeletal, soft tissue) was increased. The isolated occurrence of anal atresia and absent tail observed in one of 304 fetuses in one out of 24 litters at 250 mg/kg bw/day should be regarded as an incidental finding.
Further findings of developmental toxicity were reduced fetal weights at 250 mg/kg bw/day onwards, delayed ossification of skull bones and sternebrae and an increase in skeletal variations at 500 mg/kg bw/day onwards. There was also a very low proportion of live fetuses and an increase in the rate of soft tissue variations at 750 mg/kg bw/day.
The NOAEL for maternal toxicity and developmental toxicity was 125 mg/kg bw/day considering biologically relevant impairments in maternal and fetal body weight. The NOAEL for malformations was 250 mg/kg bw/day.

Executive summary:

In a prenatal developmental toxicity study, pregnant Sprague-Dawley rats were treated by oral gavage with aqueous NMP solutions of 0, 125, 250, 500 or 750 mg/kg bw/day during gestational days 6 through 20 (Saillenfait et al., 2002).

Significant decrements in maternal body weight gain and food consumption between treatment days 6 – 21 were observed at doses of 500 mg/kg bw/day onwards. If one considers biologically relevant effects, the maternal body weight gain was reduced by 9 % at 250 mg/kg bw/day, a reduction comparable to the statistically significant reduction in fetal body weight observed at the same dose. In addition, the net weight change was reduced by 11, 26 and 26 % at 250, 500 and 750 mg/kg bw/day, respectively.

Post implantation losses and the number of resorptions were increased at 500 mg/kg bw/day, showing a steep dose-response relationship. The rate of fetal malformations (external, skeletal, soft tissue) was increased at 500 mg/kg bw/day onwards. The principal types of malformations consisted of external (anasarca, anal atresia), soft tissue (persistent truncus arteriosus) and skeletal findings (fusion or absence of cervical arches were most prominent). The isolated occurrence of anal atresia and absent tail observed in one of 304 fetuses in one out of 24 litters at 250 mg/kg bw/day should be regarded as an incidental finding. It is not considered compound related as this type of malformation also occurs spontaneously with low frequencies in this rat strain.

Further findings of developmental toxicity were reduced fetal weights at 250 mg/kg bw/day onwards, delayed ossification of skull bones and sternebrae and an increase in skeletal variations at 500 mg/kg bw/day onwards. There was also a very low proportion of live fetuses and an increase in the rate of soft tissue variations at 750 mg/kg bw/day.

The NOAEL for maternal toxicity and developmental toxicity was 125 mg/kg bw/day considering biologically relevant impairments in maternal and fetal body weight. The NOAEL for malformations was 250 mg/kg bw/day.

It is noteworthy to mention that this assessment in respect to maternal toxicity is in contrast to the assessment of the authors that "(...) the NOAEL for maternal and developmental toxicity was 250 and 125 mg/kg/day, respectively".

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

12 May 1987

Deviations:

yes

Remarks:

two dose groups instead of three

GLP compliance:

not specified

Specific details on test material used for the study:

- colorless, light yellow
- mild amine vapor
- '100% pure NMP'

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., North Wilmington, MA, USA
- Housing: singly in suspended stainless-steel wire cages
- Diet (e.g. ad libitum): not specified
- Water (e.g. ad libitum): not specified
- Acclimation period: not specified
- Chamber atmosphere: monitored with a Wilkes Miran infrared spectrophotoemter (GC-IR with FID)
- Pregnancy: confirmed by finding sperm in the vaginal smears

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 24 (75 °F)
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

IN-LIFE DATES: From: To:

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Remarks on MMAD:

Respirable NMP aerosol was generated by syringe driving the NMP solution into nebulizers and the resulting mist was added to a stream of "make-up" air prior to entering the exposure chamber.
The particle size distribution of the aerosol was not determined.

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4*1.4 m3 stainless-steel chambers with dynamic airflow
- Method of holding animals in test chamber: not specified
- Source and rate of air: not specified
- Method of conditioning air: not specified
- System of generating particulates/aerosols: syringe driving the NMP solution into nebulizers and the resulting mist was added to a stream of "make-up" air prior to entering the exposure chamber
- Temperature in air chamber: 75°F
- Method of particle size determination: atmosphere was only monitored by a GC-IR (with FID)
- Treatment of exhaust air: Air and aerosolized NMP scrubbed throughr or with water after exiting test chamber

TEST ATMOSPHERE
- Brief description of analytical method used: Miran infrared spectrophotometer + GC (+FID) (4x 1/4-in. o.d. column containing 15% OV-101 on Chromosorb W. HP 80/100 mesh)
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: over 1 night
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

Days 6 through 15 of pregnancy

Frequency of treatment:

continuous

Duration of test:

until day 21 of pregnancy

Dose / conc.:

0.1 mg/L air (nominal)

Remarks:

concentration calculated from amount (mg) of NMP in the impinger liquid and the amount of chamber air passed through the impinger

Dose / conc.:

0.36 mg/L air (nominal)

Remarks:

concentration calculated from amount (mg) of NMP in the impinger liquid and the amount of chamber air passed through the impinger

No. of animals per sex per dose:

25

Control animals:

yes, concurrent no treatment

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: not specified

DETAILED CLINICAL OBSERVATIONS: No data (may be included into cage side observations)

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly and on the day of laparotomy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: uterus, ovaries; other tissues/organs examined grossly

OTHER:
- skeletal, visceral and neural abnormalities in fetuses

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of late resorptions: Yes
- Number of partial resorptions: Yes
- Number of early resorptions: Yes

Fetal examinations:

- External examinations: Yes: not specified per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Statistics:

Fisher's exact test (Siegel, 1956): incidence of resorptions and abnormalities among the litters
ANOVA and the least-significance-difference test (Steel and Torrie, 1960): Maternal and fetal body weights and body measurements
Mann-Whitney U test (Siegel, 1956): numbers of corpora lutea, implantations, and live fetuses

Clinical signs:

no effects observed

Description (incidence and severity):

- sporadic lethargy and irregular respiration were found in several rats at both the 0.36 and 0.1 mg/liter during the first 3 days of exposure; effects not seen thereafter during the 10-day recovery period

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no difference to control animals

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- no differences compared to control animals

Gross pathological findings:

no effects observed

Description (incidence and severity):

- no significant pathological changes in any vital organs or tissues observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- no significant pathological changes compared to control animals

Histopathological findings: neoplastic:

not specified

Details on results:

Results are given in a summarized form in table 1.

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

- at 0.1 mg/l: 2/10 total resorption litter losses
- at 0.36 mg/l: no loss

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

- at 0.1 mg/l: number of resorptions per litter was significantly lower
- at 0.36 mg/l: similiar to control group

Dead fetuses:

no effects observed

Description (incidence and severity):

- no differences compared to control animals

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

- five of twenty-five females in the 0.1 mg/liter exposure group were not impregnated
- two of twenty-five females in the 0.36 mg/liter exposure group were not impregnated

Details on maternal toxic effects:

No maternal toxicity was found up to the highest concentration.
Results are given in a summarized form in table 1.

Dose descriptor:

NOAEC

Effect level:

0.36 mg/L air (nominal)

Based on:

test mat.

Remarks on result:

other: no adverse effects observed

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - at 0.1 mg/l: significantly higher
- at 0.36 mg/l: similiar to control group

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not examined

External malformations:

no effects observed

Description (incidence and severity):

- no differences compared to control animals

Skeletal malformations:

no effects observed

Description (incidence and severity):

- no differences compared to control animals

Visceral malformations:

no effects observed

Description (incidence and severity):

- no differences compared to control animals

Details on embryotoxic / teratogenic effects:

No abnormal development was detected in the vital organs and skeletons of the fetuses.
Results are given in a summarized form in table 1.

Dose descriptor:

NOAEC

Effect level:

0.36 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effect observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Lowest effective dose / conc.:

0.36 mg/L air (nominal)

Treatment related:

no

Table 1: Effects of NMP on the maternal toxicity, the outcome of pregnancy and the fetal development

 

Endpoint	0 mg/L	0.10 mg/L	0.36 mg/L
	0 ppm	24 ppm	87 ppm
Females bred [n]	25	25	25
Pregnant dams [n]	22	20	23
Litters with early resorption	9(40.9%)	10 (50.0%)	10 (43.5%)
Litters with late resorption	1 (4.5%)	3 (15%)	0
Litters with partial resorptions	10(45.5%)	10 (50.0%)	10(43.5%)
Litters with total resorption	0	2 (10.0%)	0
Body weight^a(g)
Initial body weight of pregnant females	227.6 ± 23.6	232.5 ± 29.5	224.6 ± 17.8
Final body weight of pregnant females	360.9 ± 53.0	359.0 ± 45.5	369.8 ± 38.1
Fetal examination^a
Corpora lutea/litter [n]	14.3 ± 3.4	11.0 ± 4.0*	13.8 ± 2.6
Implantations/litter [n]	9.5 ± 3.8	8.2 ± 4.4	9.6 ± 3.5
Live fetuses / litter, mean [n]	8.7 ± 3.7	7.4 ± 4.4	8.5 ± 3.5
Fetal weight (g)	4.0 ± 0.4	4.4 ± 0.4**	4.0 ± 0.3
Number of litters (fetuses) examined	22 (191)	18(149)	23 (196)
Gross
Small subcutaneous hematomas	5 (8)	6 (8)	6 (7)
Petechial hemorrhage	9 (9)	2 (2)*	7 (13)
Skeletal
Delayed ossification	14 (32)	8 (13)	19 (55)
14th rudimentary rib(s)	18 (51)	13 (34)	19 (55)
Wavy ribs	7 (11)	1 (1)*	1 (3)*

BW = body weight

a Values are expressed as means ± SD.

*,** Significant differences from the control (air), P<0.05 (lower) and P<0.05 (higher), respectively.

Conclusions:

Exposure during pregnancy from day 6 to day 15 of gestation did not affect either the outcome of pregnancy or embryonal growth rate in rats. No abnormal development was detected in the vital organs and skeletons of the fetuses. The NOAEC observed in this study is 0.36 mg/l which is equivalent to 360 mg/m3 (87 ppm).

Executive summary:

Pregnant Charles River CD Sprague-Dawley rats were exposed to N-methyl-2-pyrrolidone (NMP) at atmospheric concentrations of 0.1 and 0.36 mg/liter for 6 hr/day on pregnancy days 6 through 15 of gestation (Lee et al., 1987). Except for sporadic lethargy and irregular respiration in several rats the first 3 days of exposure, there were neither abnormal clinical signs nor pathological lesions in the maternal rats. Exposure did not affect either the outcome of pregnancy or embryonal growth rate. No abnormal development was detected in the vital organs and skeletons of the fetuses. The NOAEC observed in this study is 0.36 mg/l which is equivalent to 360 mg/m3 (87 ppm).

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: > 99.5 %
- Source: Merck, Darmstadt, Germany

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories, Saint-Germain-sur-l'Arbresle, France
- Nulliparous, not pregnant: yes
- Age at study initiation: young adult
- Weight at study initiation: 180 - 200 g (females)
- Housing:
- before exposure: single housing, clear polycarbonate cages with stainless-steel wire lids and corn cob granules as bedding
- during exposure: stainless-steel wire mesh exposure cages in 200 L stainless-steel inhalation chambers with dynamic and adjustable air flow (6 - 10 m³/h)
- Diet: commercial diet (pellets, UAR A limentation, Villemoisson, France), ad libitum
- Water: drinking water, ad libitum
- Food and water were withheld during exposures.
- Acclimation period: about 2 weeks


ENVIRONMENTAL CONDITIONS
Before Exposure
- Temperature (°C): 21 ± 1
- Humidity (%): 50 ± 5
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours

During Exposure
- Temperature (°C): 23 ± 0.6
- Humidity (%): 40 ± 5.4
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless-steel wire mesh exposure cages in 200 L stainless-steel inhalation chambers
- Method of holding animals in test chamber: cage
- Source and rate of air: compressed air, 10 - 30 L/min
- System of generating vapour:
- infusion pump to deliver a constant rate of liquid NMP to the top of a heated (95 °C) glass column filled with glass beads
- compressed air (10 - 30 L/min) heated by a gas heater introduced at the bottom of the glass column in a counter current fashion to liquid NMP
- Temperature, humidity, pressure in air chamber: 23 ± 0.6 °C, 40 ± 5.4 %, the chambers were maintained at a negative pressure of no more than 3 mm water gauge
- Method of particle size determination: GC-FID

TEST ATMOSPHERE
- Brief description of analytical method used: Concentrations were monitored with GC-FID; exposure concentrations were determined once during each 6-hour exposure. The presence of liquid particles was examined at the high concentration only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of NMP were monitored continuously with a gas chromatograph (Intersmat IGC 120 FB) equipped with a flame ionization detector and an automatic gas sampling valve.
As NMP has a low vapour pressure, the presence of liquid particles was evaluated at the highest concentration evaluated (i.e. 120 ppm) with an optical particle counter (Grimm 105, Grimm Labortechnik, Ainring, Germany).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male/2 or 3 females
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: yes, adult males from the same strain and supplier
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation

Duration of treatment / exposure:

day 6 through day 20 of gestation

Frequency of treatment:

6 hours/day

Duration of test:

21 days

Dose / conc.:

30 ppm (analytical)

Remarks:

(± 1 ppm); equal to 124 mg/m3

Dose / conc.:

60 ppm (analytical)

Remarks:

(± 2 ppm); equal to 247 mg/m3

Dose / conc.:

121 ppm (nominal)

Remarks:

(± 4 ppm); equal to 494 mg/m3

No. of animals per sex per dose:

25-26 rats (26 only highes dose)
(20-25 pregnant rats)

Control animals:

yes, concurrent no treatment

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0, 6, 13 and 21

FOOD CONSUMPTION: Yes
- Time schedule: measured for the gestation day intervals 6-13 and 13-21

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: ovaries and uterus (incl. weighing), gross necropsy

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Other: observation of dead fetuses

Fetal examinations:

- External examinations incl. sex : Yes: all per litter, including the oral cavity
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

ANOVA and Dunnett's test: corpora lutea, implantation sites, live fetuses, body weight
Kruskal-Wallis test followed by Mann-Whitney test: post-implantation loss, dead fetuses, resorption, alteration among litter
Fisher's test: pregnancy rate, incidences of fetal alterations
- The litter was used as the basis for the analysis of fetal variables.

Indices:

None

Historical control data:

None

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

All animals survived the exposure.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- reductions in maternal body weight gain on GD 6-13 (P<0.01) and 6-21 (P<0 .05) for 60, 120 ppm

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- reduction in food intake on GD 6-13, 13-21 and 6-21 (P <0.05) for 60, 120 ppm

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Table 1 gives an overview of main results.

Number of abortions:

no effects observed

Description (incidence and severity):

- mean numbers of non-live implants and resorptions were comparable across groups

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- mean numbers of implantation sites were comparable across groups

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

- mean numbers of non-live implants and resorptions were comparable across groups

Early or late resorptions:

no effects observed

Description (incidence and severity):

- mean numbers of non-live implants and resorptions were comparable across groups

Dead fetuses:

no effects observed

Description (incidence and severity):

- number of live fetuses were comparable across groups

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxicity was observed at 60 and 120 ppm, as indicated by reductions in body weight gain during the first half of exposure.
Table 1 gives an overview of main results.

Dose descriptor:

NOAEC

Effect level:

60 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: The effects for maternal toxicity were observed at higher concentrations and appear to follow a dose-response relationship. Therefore, the effects seen at 60 ppm are taken into account for the derivation of the NOAEC.

Dose descriptor:

LOAEC

Effect level:

120 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: The effects for maternal toxicity were observed at higher concentrations and appear to follow a dose-response relationship. Therefore, the effects seen at 60 ppm are taken into account for the derivation of the NOAEC.

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- concentration-related decrease in fetal body weights (all, males, and females, P< 0.01), which achieved statistical significance at 120 ppm (5-6% lower than control)
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - as discussed above

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- mean number of live fetuses were comparable across groups

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- no changes compared to control group

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

Several common external, visceral and skeletal variations were observed, with no indication ofany adverse effects related to NMP exposure.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Several common external, visceral and skeletal variations were observed, with no indication ofany adverse effects related to NMP exposure.

Visceral malformations:

no effects observed

Description (incidence and severity):

Several common external, visceral and skeletal variations were observed, with no indication ofany adverse effects related to NMP exposure.

Details on embryotoxic / teratogenic effects:

Only mean fetal weights were reduced at 120 ppm. There was no difference in the incidences of external, visceral and skeletal malformations or variations between the control and the exposure groups.
Table 1 gives an overview of main results.

Dose descriptor:

NOAEC

Effect level:

60 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Dose descriptor:

LOAEC

Effect level:

120 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

no effects observed

Developmental effects observed:

yes

Lowest effective dose / conc.:

120 ppm (analytical)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Table 1: Results from the maternal and fetus effects.

 

Endpoint	0 ppm	30 ppm	60 ppm	120 ppm
	0 mg/m^3	124 mg/m^3	247 mg/m^3	494 mg/m^3
Females mated [n]	25	25	25	26
Pregnant dams [n]	24	20	20	25
Dams with viable fetuses [n]	24	20	19	25
Body weight^a
BW (g) day 0	235 ± 18	235 ± 19	243 ± 20	237 ± 24
BW change (g) day 0-6	35 ± 11	33 ± 8	30 ± 9	32 ± 10
BW change (g) day 6-13	31 ± 7	27 ± 9	25 ± 8 *	23 ± 7 **
BW change (g) day 13-21	104 ± 22	95 ± 31	96 ± 32	89 ± 22
BW change (g) day 6-21	134 ± 27	122 ± 36	122 ± 36	112 ± 25
Absolute BW gain (g)^b	32 ± 9	28 ± 10	26 ± 11	26 ± 10
Food consumption (g/day)
Day 0-6	22 ± 2	22 ± 2	22 ± 2	22 ± 2
Day 6-13	23 ± 2	22 ± 1	22 ± 2	21 ± 2
Day 13-21	26 ± 2	24 ± 2	25 ± 3	24 ± 2 *
Day 6-21	25 ± 2	23 ± 2	23 ± 2	23 ± 2 *
Fetal examination
Live fetuses / litter, mean [n]	13.9 ± 3.8	12.6 ± 4.7	14.0 ± 3.4	12.0 ± 4.1
Fetal weights, males, mean [g]	5.81 ± 0.39	5.74± 0.32	5.64 ± 0.21	5.51 ± 0.44*
Fetal weights, females, mean [g]	5.54 ± 0.37	5.42 ± 0.47	5.32 ± 0.30	5.21 ± 0.44*
Fetuses with external var. [%]	0.6 ± 2.2	0.7 ± 2.1	0.6 ± 1.8	0.5 ± 1.7
Fetuses with visceral var. [%]	0.7 ± 3.5	2.6 ± 5.4	0.8 ± 3.3	0
Fetuses with skeletal var. [%]	26.6 ± 25.9	19.8 ± 15.6	18.3 ± 12.0	15.5 ± 24.1

BW = body weight

a Values are expressed as means ± SD.

b Body weight gain during GD 6–21 minus gravid uterine weight.

*,** Significant differences from the control (air), P<0.05 and P<0.01, respectively.

Conclusions:

The results of this study indicate that inhalation exposure of rats to NMP vapours during the embryonic and fetal period did not result in selective toxicity to the offspring. Maternal toxicity was observed at 60 and 120 ppm (124 and 247 mg/m3), as indicated by reductions in body weight gain during the first half of exposure. This effect was transient and no significant decrease was found after deduction of the gravid uterus weight from the overall weight gain during pregnancy. The dams also exhibited a slight reduction in food consumption at the high concentration. Fetal toxicity limited to a decrease in fetal weight occurred at 120 ppm (494 mg/m3). Thus, the NOAELs for maternal and developmental toxicity are 30 and 60 ppm (124 and 247 mg/m3), respectively.

Executive summary:

The developmental toxicity of inhaled N-methyl-2-pyrrolidone (NMP) was studied in Sprague-Dawley rats. Pregnant rats were exposed whole body to NMP vapours at concentrations of 0, 30, 60 and 120 ppm (124, 247 and 494 mg/m³), 6 h/day, on gestational days (GD) 6 through 20 (Saillenfait, 2003). Maternal body weight gain was significantly decreased at 60 and 120 ppm on GD 6-13 and maternal food consumption was reduced at 120 ppm on GD 13-21 . No significant difference in the gestational weight change corrected for the weight of the gravid uterus was observed, whatever NMP concentration. There were no adverse effects on embryo/fetal viability or evidence of teratogenicity at any concentration tested. Fetal toxicity indicated by reduced fetal weight was observed at 120 ppm (494 mg/m³). Thus, the no-observed-adverse-effect concentration (NOAEC) for maternal and developmental toxicity was 30 and 60 ppm, respectively (124 and 247 mg/m³).

Reference 5

Endpoint:

developmental toxicity

Remarks:

within a 2-generation-study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

not specified

Deviations:

yes

Remarks:

only one concentration tested; part of a 2-generation study

Principles of method if other than guideline:

segment of a reproduction 2-generation toxicity study

GLP compliance:

yes

Specific details on test material used for the study:

- Source: E. I. du Pont De Nemours and Company, Richmond, Virginia, USA
- Purity: 99.9%
Impurities:
- 1.5 or isomer-dimethyl-2-pyrrolidone (986 ppm)
- N-methyl-succinimide (251 ppm)
- 2-pyrrolidinone (14 ppm)
- low boiling impurities (36 ppm), high boiling impurities (40 ppm)

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding North Wilmington, MA, USA
- Age at study initiation:
- first group (P0 generation): 21 days
- second group (unexposed mates for F1 generation): 64 days
- Weight at study initiation:
- first group: 32-50 g
- second group: 165-217 g
- Fasting period before study: not specified
- Housing:
- before exposure: individually, cages made of stainless steel wiremesh
- during exposure: individually, stainless steel cages placed within 750 or 900 liter glass and stainless steel chambers
- animal positions within the chambers were rotated daily
- Diet: ad libitum except during the inhalation exposures; Purina Certified Rodent Chow No. 5002 (Ralston Purina Company, St. Louis, MO, USA)
- Water: ad libitum, except during the inhalation exposures
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (72 ± 2°F)
- Humidity (%): 40-60
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: nitrogen carried vapors and was mixed with dilution air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: NMP pumped through inclined tube furnace heated to 195-210°C, N2 carried vapours throught the tube
- Method of holding animals in test chamber: hold in cages
- Source and rate of air: dilution air, 700 L/min
- Temperature, humidity, oxygen concentration: 18.9 - 27.7 °C (within control animal chambers: 19.2 - 28.3°C), about 50% r.h., below 21%
- Method of oxygen monitoring: Biomarine Oxygen Analyzer (Grade 1225R)
- Treatment of exhaust air: ventilation from bottom chamber through aqueous scrubbing tower

TEST ATMOSPHERE
- Brief description of analytical method used: HP 5710A GC with nitrogen/phosphorus detector
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test atmosphere measured with HP 5710A GC with nitrogen/phosphorus detector.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: 5 days
- After 5 days of unsuccessful pairing replacement of first male by another male with proven fertilit of same treatment group.
- Further matings after two unsuccessful attempts: yes,if not mated female placed with another male of same treatment group for 5 d
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal lavage referred to as day 0 of pregnancy

Duration of treatment / exposure:

males: 100 days, females: 106-110 d

Frequency of treatment:

6 h/d,7d/week

Duration of test:

minimum of 14 weeks

Dose / conc.:

116.4 ppm (analytical)

Remarks:

equal to 478 mg/m3

No. of animals per sex per dose:

10 males and 20 females per dose level

Control animals:

yes, concurrent no treatment

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- P0 upon arrival and twice more before exposure
- during exposure while in the chamber and during handling

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
- druing handling (remove from chamber and return into chamber)
- P0 males: weekly for recovery animals
- mated females: d1-d21 gestation
- dams: pathological changes

BODY WEIGHT: Yes
- Time schedule for examinations:
- P0 upon arrival and twice more before exposure
- P0 males weekly until euthanization; females weekly until mating and on d1 and d21 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: liver, uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter, except for the heads fixed in Bouin's solution
- Head and eyelid examinations: Yes: all per litter; heads fixed in Bouin's solution

Statistics:

- One way ANOVA, significant F-value resulted in Dunnett's test, nonparametric procedures resulted in Batlett's test: body weight, body weight gain, organ weight and gestational length
- Cochran-Armitage test for trend, if significant Fisher's exact followed: incidence of clinical observations
- Fisher's exact: mating, fertility, gestation indices, and for litter survival
- Jonckheere's test, two-tailed Mann-Whitney U test: Live fetuses, dead fetuses, resorptions, percent resorptions, corpora lutea, fetal weight and incidence of alterations
- test for trend and pairwise test between control and experimental groups, Cochran-Armitage and Fisher's exact test: incidence of pregnancy, clinical signs, and matemal death
- two-way ANOVA: differences among groups

SIGNIFANCE LEVEL
- alpha = 0.05 for all, except for Bartlett's test (alpha = 0.005)

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three P0 rats died during the study; two in the control group and one in the 51 ppm group. One of the two control rats died from an injury received during handling.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Food efficiency:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

An exposure-related reduction in response to sound was noted at 116 ppm, but not at either 10 or 51 ppm.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Dead fetuses:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): - no effects compared to control animals observed

Dose descriptor:

NOAEC

Effect level:

116 ppm

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- slight but statistically significant decrease in fetal body weight was observed at 116 ppm (7% = 3.37 g versus 3.62 g in controls; p<0.025).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

External malformations:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Visceral malformations:

no effects observed

Description (incidence and severity):

- no effects compared to control animals observed

Details on embryotoxic / teratogenic effects:

No significant difference in the incidence of fetal malformations, variations or retarded development was observed in the exposed rats compared to the control.
An exposure-related, slight but statistically significant decrease in fetal body weight was observed at 116 ppm (7% = 3.37 g versus 3.62 g in controls; p<0.025).
Details on study results are given in table 1.

Dose descriptor:

LOAEC

Effect level:

116 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

no effects observed

Developmental effects observed:

yes

Lowest effective dose / conc.:

116 ppm (analytical)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

not specified

Table 1: Combined results of the P0 generation of the combined 2-year-generation study and the developmental toxicity study. Groups CI and EIII for use in the developmental toxicity phase of the study.

Group	CI/II	EI	EII	EIII	EIV	EV
Exposure Level (ppm)	0	10	51	116	0	116
	0	10	51	116	116	0
Mean Number of Offspring/Litter
Born	13.7	14.0	13.7	14.2	13.9	14.7
Born Alive	13.5	13.9	13.5	14.1	13.9	14.6
Day 4 PP Preculling	13.4	13.9	13.3	13.9	13.6	14.3
Day 4 PP Postculling	8.0	7.9	7.9	8.0	8.0	7.9
Day 14 PP	8.0	7.9	7.9	8.0	8.0	7.9
Day 21 PP	8.0	7.8	7.9	8.0	8.0	7.9
Viability Index(%)	99.3	100.0	98.6	98.3	98.0	97.7
Lactation Index(%)	100.0	98.4	99.0	100.0	100.0	100.0
Sex Ratio (% Males)	0.53	0.46^a	0.43	0.46	0.48	0.50
Offspring Weight/ Litter (g)
Day 1 PP	7.5	7.0^a	7.1	6.7^a	7.1	7.3
Day 4 PP Preculling	10.8	10.0^a	l0.3	9.6^a	10.1	10.5
Day 4 PP Postculling	10.7	9.9^a	10.2	9.6^a	9.9^a	10.6
Day 14 PP	30.8	27.8^a	29.5	28.7^a	28.6^a	32.0
Day 21 PP	49.1	45.6^a	47.4	46.9^a	47.2	51.6^a
^a= Significantly different from Control Group, p ≤ 0.05 C: control group; E: exposure group

Conclusions:

The NOAEC for maternal toxicity was 116 ppm (equivalent to 478 mg/m3) with exposure exclusively in vapor form.
Based on the reduced pup body weights the investigated concentration of 116 ppm was not a NOAEC for fetotoxicity, however, in respect to the small effect, this concentration should be considered as LOAEC for developmental toxicity.

Executive summary:

As part of a reproduction toxicity study (Solomon et al., 1995), 10 male and 20 female Sprague-Dawley rats were exposed to 0 or 116 ppm (478 mg/m³) NMP vapor starting at 34 days of age for 6h/d. For the male rats, exposure continued until day 134 of age, the end of the mating period. The females were exposed to gestation day 20 (day 140 -144). Body weights of the dams were recorded on gestation day 1 and 21, and clinical signs were monitored daily before, during and after exposure. After sacrifice, dams were examined for gross pathological changes, livers were weighed, corpora lutea were counted and resorptions were determined by staining. The fetuses were sexed and subjected to external, visceral, and skeletal examinations. The indices of reproductive performance for the NMP-exposed rats did not differ significantly from those obtained for the control rats. Rats exposed to 116 ppm (478 mg/m³) had a detectable decrease in response to sound. No other signs of NMP-related toxicity were detected among the parental rats. An exposure-related slight decrease in pup weight was detected at 116 ppm. The LOAEC for developmental toxicity was therefore 116 ppm.

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-06-02 to 1991-07-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Paris 1981

Deviations:

no

GLP compliance:

yes

Remarks:

in accordance with OECD Laboratory Practice Paris, 1981

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, from continuous production, tank No. 53
- Analytical purity: 99.8 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: under N2, to be protected from air
- Stability under test conditions: stability ensured by substance supplier

Species:

rabbit

Strain:

Himalayan

Remarks:

Chbb:HM (outbread strain)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. K. Thomae GmbH, Biberach/Riss, (D-W7950) Germany
- Age at study initiation: 23 - 30 weeks old
- Weight at study initiation: approximately 2,424 g (mean)
- Housing: during the period when the rabbits were not exposed they were housed singly in wire cages (type K 300/8, EBECO, Becker & Co., Castrop-Rauxel, FRG). During exposure: singly, glass steel inhalation chambers, in modified fixation cages wrapped with aluminum foil to avoid fur contamination.
- Diet: commercial diet (Kliba rabbit laboratory diet, 24-341-4, 10 mm pellets, Klingenthalmühle AG, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: yes, from days 1-6 p.i. (post insemination), animals were exposed to fresh air over 6 h/d in inhalation-chambers to adapt to the exposure conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

IN-LIFE DATES: From: 03 June 1991 To: 04 July 1991

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Remarks:

compressed air for aerosol mixing

Mass median aerodynamic diameter (MMAD):

>= 2.7 - <= 3.5 µm

Geometric standard deviation (GSD):

3

Remarks on MMAD:

- only two independet measurements were performed

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass-steel inhalation chamber (BASF AG, V: approx 1400 L)
- Method of holding animals in test chamber: wrapped with aluminium foil in modified fixation cages
- Source and rate of air: compressed air with 25-28 m³/hour, conditioned supply air with 3 m³/hour
- System of generating particulates/aerosols: The test substance was supplied to a two-component atomizer by means of a continuous metering pump. By means of compressed air the aerosol was generated into an aerosol mixing vessel. In the mixing vessel the aerosol was mixed with conditioned supply air and passed through a cyclonic separator into the inhalation chamber.
- Temperature, humidity, pressure in air chamber: continuously measured and partially regulated.
- Method of particle size determination: gas chromatography after absorption of NMP measured samples in 2-propanol.

TEST ATMOSPHERE
- Brief description of analytical method used: a 50 mL graduated flask was filled up to the calibration mark with 2-propanol after 1 mL of internal standard (internal standard = C16KW) was added to the samples. Then 2 mL glass tubes were filled for the automatic sampler (automatic metering of the samples for the gas chromatograph) and analyzed. The injected volume was 1 µL.
Mass values NMP were obtained from the appropriate area integrals. The concentration values NMP of the test groups were calculated from these taking the sample volumes and the injected volume into account.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations were monitored with a Hewlett-Packard 5840 A with an automatic sampler 7671 A. Particle size analysis was performed with a cascade impactor at a sampling velocity of 1.25 m/s.

Details on mating procedure:

- Acclimatization period: 5 days
- Impregnation procedure: artificial insemination
- Verification of same strain and source of both sexes: yes, the pooled ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females.
- Day of insemination was designated as day 0 (beginning of the study), following day = day 1 post implantation

Duration of treatment / exposure:

day 7 through day 19 of gestation

Frequency of treatment:

6 hours/day

Duration of test:

29 days (including a post-exposure observation period: day 20 p.i. to the day of sacrifice)

Dose / conc.:

0.2 mg/L air (nominal)

Remarks:

equivalent to 49 ppm

Dose / conc.:

0.5 mg/L air (nominal)

Remarks:

equivalent to 122 ppm

Dose / conc.:

1 mg/L air (nominal)

Remarks:

equivalent to 243 ppm

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: the concentrations for this study were fixed depending on available toxicity data and the results of an inhalation pretest of the prenatal and maternal toxicity with concentrations of 0, 0.3, 1 and 2 mg/L (Project No. 21R0544/90049).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: on workdays at least 3 times on exposure days and, as a rule, once during the preflow period and the post-exposure observation period.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 (= day of insemination) and on days 3, 7, 10, 13, 16, 19, 21, 24, 27 and 29 p .i .

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 p.i.
- Organs examined: ovaries and uterus, gross necropsy

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: dead fetuses

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: [if heads revealed severe findings]

Statistics:

Dunnett's test (body weight, body weight change, corrected body weight gain, uterus weight, fetal and placental weights, corpora lutea, implantations, pre- post implantation losses, resorptions, live fetuses)
Fisher's exact test (conception rate, mortality of does, fetal findings)

Indices:

Conception rate (%), preimplantation loss (%), postimplantation loss (%)

Historical control data:

Historical control data were included in the report.

Clinical signs:

no effects observed

Description (incidence and severity):

- no abnormal clinical signs or findings occurred

Mortality:

no mortality observed

Description (incidence):

- no deaths were recorded

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no influence in a statistically significant manner compared to the control animals
- body weight gain on day 29 p. i. minus body weight on day 7 p. i. do not show any differences of biological relevance

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- yellowish-orange urine discoloured the bedding in all test groups, probably due to a metabolic compound of NMP

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- no substantial differences concerning the uterus weights between controls and the test groups

Gross pathological findings:

no effects observed

Description (incidence and severity):

- some spontaneous necropsy findings at necropsy of the dams were found in all dose groups

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

No clinical signs of maternal toxicity occurred during the study. A concentration and exposure time dependent yellowish-orange discoloration of urine and bedding probably by a metabolic compound of NMP showed the systemic availability of the substance.
With respect to the pretest (Proj. No. 21R0544/90049, see selection of concentrations) it is assumed that the level of maternal toxicity was reached.

Number of abortions:

no effects observed

Description (incidence and severity):

- no substance-related and/or statistically significant differences

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- no substance-related and/or statistically significant differences

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

- no substance-related and/or statistically significant differences

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

- conception rate = 100% in all groups

Details on maternal toxic effects:

Maternal toxicity was noted in a preceding dose range-finding study at 1.0 and 2.0 mg/L in form of increased liver weights and impaired clinical chemistry parameters (BASF AG, Department of Toxicology (1991), unpublished results, Proj. No. 21R0544/90049).

In the main study, no clinical signs of toxicity were observed in the does in any group; yellowish-orange urine was observed in a concentration- and exposure time-dependent pattern. Body weights, body weight changes, and corrected body weight gains were equivalent across all groups. No does died on study. There were no treatment-related findings in the does at scheduled necropsy on gestation days 29. All does were pregnant in all groups, and uterine weights were equivalent across all groups. There were no effects of treatment on pre- or post-implantation loss or on the number of resorptions, dead, or live fetuses per litter.

Dose descriptor:

NOAEC

Effect level:

0.5 mg/L air

Based on:

test mat.

Basis for effect level:

other: maternal toxicity in the dose-range finder study

Remarks on result:

other: equivalent to 122 ppm

Dose descriptor:

LOAEC

Effect level:

1 mg/L air

Based on:

test mat.

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Remarks on result:

other: equivalent to 243 ppm

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

- mean fetal weights were not influenced after inhalation of the test substance
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- sex distribution was comparable among all test groups

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- external malformations, i.e. meningoeele, shortened toes were comparable to the historical control animals at a comparable frequency
- no so-caIled unclassified observations (like placentae fused) in any fetus

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

- occurrence of accessory 13th rib(s) was clearly increased in the high dose group fetuses, which might be retated to the exposure of the test substance
- relevant values (fetal incidence: 32%; Iitter incidence: 80%) are above the highest values of the historical control group fetal incidence: 2.4% (0.0 - 8.4%); Iitter incidence: 10.8% (0.0 - 40.0%), as depicted in table 3

- in one control fetus: absent lumar vertebra
- in one 0.5 mg/L: cervieal vertebrae fused and/or of irregular shape
- in one 1.0 mg/L: ectrodactyly, this fetus showed shortened toes already during the external examination
- variations occurred without a clear dose-response relationship and/or without any biologically relevant, statisticatly significant differences between the groups
- retardations found in all groups but occurred without any biological relevant differences between the groups; a spontaneous nature is suggested for the low number of total skeletal retardations in the 0.5 mg/L group

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- soft tissue malformations, i.e. to the heart (septal defect, dilatation of Ieft ventricle) or the great vessels (incl. aorta), agenesia of gallbladder were comparable to the historical control animals at a comparable frequency
- one fetus each of the control- and the low concentration group and two fetuses of the 0.5 mg/I group showed so-called unclassified observations (focal liver necrosis or blood coagulum around bladder)

Details on embryotoxic / teratogenic effects:

The high concentration (test group 3, 1.0 mg/L) led to an increased occurrence of one skeletal variation (accessory 13th rib(s)). The other concentrations (0.5 and 0.2 mg/L) elicited no adverse effects on the fetuses. The distinctly increased number of fetuses with accessory 13th rib(s) in the high concentration is assessed as a non-specific embryotoxic effect at a maternally toxic concentration.

Dose descriptor:

NOAEC

Effect level:

0.5 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Significantly increased incidence of skeletal variations (accessory 13th rib(s)) at 1.0 mg/L.

Remarks on result:

other: equivalent to 122 ppm

Dose descriptor:

LOAEC

Effect level:

1 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Significantly iIncreased incidence of skeletal variations (accessory 13th rib(s)) at 1.0 mg/L.

Remarks on result:

other: equivalent to 243 ppm

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

Description (incidence and severity):

- relevant values (fetal incidence: 32%; Iitter incidence: 80%) are above the highest values of the historical control group fetal incidence: 2.4% (0.0 - 8.4%); Iitter incidence: 10.8% (0.0 - 40.0%)

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 mg/L air (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

not specified

Analysis of atmosphere: analyzed concentrations correspond to nominal concentrations. Particle size measurements showed the presence of aerosols at 0.5 and 1.0 mg/L with MMADs and respirable fractions of 2.7 µm and 90.6 % or 3.5 µm and 88.8 %.

Table 1: Results of particle size analysis and assumed amount of particles reaching the lung.

ANALYSIS OF ATMOSPHERE
Endpoint	Dose Group [mg/L]
	0	0.2	0.5	1.0
Aerosol
MMAD [µm]	-	-	2.7	3.5
Respirable fraction [%]	-	-	90.6	88.8
-: no aerosol, MMAD: mass median aerodynamic diameter

 

Table 2: Results of the observations and findings of the dams.

MATERNAL DATA
Endpoint	Dose Group [mg/L]
	0	0.2	0.5	1.0
Mated females on study [n]	15	15	15	15
Pregnant females [n]	15	15	15	15
Mortality of dams	0	0	0	0
Clinical symptoms	-	-	-	-
Body weight gain (days 7-19) [g]	67.7	40.3	64.3	40.5
Corr. body weight gain [g]	-88.5	-87.4	-92.8	-115.8
-: no effect, Corr.: corrected

Table 3: Terratogenic and developmental results observed in dams and pups.

CAESARIAN SECTION / FETAL EXAMINATION
Endpoint	Dose Group [mg/L]
	0	0.2	0.5	1.0
Females mated [n]	15	15	15	15
Pregnant dams [n]	15	15	15	15
Aborted [n]	0	0	0	0
Premature birth [n]	0	0	0	0
Dams with viable fetuses [n]	15	14	15	15
Dams with all resorptions [n]	0	1	0	0
Female mortality [n]	0	0	0	0
Uterus weight, mean [g]	340.5	297.3	328.9	329.6
Corpora lutea, mean [n]	7.9	7.7	8.3	7.8
Implantation sites, mean [n]	6.7	6.1	6.9	7.3
Live fetuses, mean [n]	6.5	6.1	6.3	6.5
Total resorptions, mean [n]	0.2	0.5	0.5	0.8
Early resorptions, mean [n]	0.1	0.3	0.3	0.4
Late resorptions, mean [n]	0.1	0.1	0.2	0.4
Dead fetuses	0	0	0	0
Placental weights, mean [g]	4.4	4.3	4.4	4.0
Fetal weights, males, mean [g]	39.2	38.9	38.4	38.2
Fetal weights, females, mean [g]	39.4	38.9	38.9	38.3
Fetal skeletal variations, fetal incidence
Accessory 13th rib(s) [%]	6.2	5.9	11	32**
Total skeletal variations [%]	14	9.4	19	34**
All classified fetal observations, fetal incidence
Total malformations [%]	2.1	2.4	3.2	1.0
Total variations [%]	41	28	39	48
Total retardations [%]	52	40	25**	38
-: no effect, Corr.: corrected, **: p < 0.01

Conclusions:

No clinical signs of maternal toxicity occurred during the study. A concentration and exposure time dependent yellowish-orange discoloration of urine and bedding probably by a metabolic compound of NMP shows the systemic availability of the substance.
With respect to the pretest (Proj. No. 21R0544/90049, see selection of concentrations) it is assumed that the level of maternal toxicity was reached.

The high concentration (test group 3, 1.0 mg/L led to an increased occurrence of one skeletal variation (accessory 13th rib(s)). The other concentrations (0.5 and 0.2 mg/L) elicited no adverse effects on the fetuses. The distinctly increased number of fetuses with accessory 13th rib(s) in the high concentration is assessed as a non-specific embryotoxic effect at a maternally toxic concentration.

No teratogenic effect could be found.

Executive summary:

Groups of 15 inseminated Himalayan rabbits were inhalation exposed to NMP concentrations of 0, 0.2, 0.5 and 1.0 mg/L (0, 49, 122, 243 ppm, vapor and vapor-aerosol mixture) for 6 hours daily on gestation day 7 through 19 (BASF, 1991).

The animals were treated in whole body inhalation chambers, sitting in specially shielded cages to avoid contamination of body surface (head-nose exposure).

No mortality occurred and no signs of maternal toxicity were noted in the examined parameters (clinical findings, body weight, body weight gain, corrected body weight, gross pathology) at any concentration.

However, a range-finding study (BASF SE, 21R0544/90049, 1991), which examined a wider range of parameters, showed maternal toxicity at 1.0 and 2.0 mg/L in form of increased liver weights and impaired clinical chemistry parameters. This concentration showed also slight fetotoxicity due to an increased incidence of supernumerary 13th ribs as a further indication of non-specific maternal stress and could be an evidence that a minimal toxic dose had been achieved.

The NOAEC for maternal toxicity and for developmental toxicity was 0.5 mg/L (122 ppm).

Reference 7

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 99.97 %
- Lot/batch No.: 821
- Source: GAF Corp., Wayne, NJ, USA

Species:

rat

Strain:

Sprague-Dawley

Remarks:

BLU:(SD)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farm Inc., Altamont, NY, USA
- Age at study initiation: young adult
- Housing: singly, wire-mesh bottom cages in temperature controlled quarters
- Diet: commercial diet (Ground Charles River rat/mouse/hamster formula, Agway), ad libitum
- Water: fresh tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: 25 cm², rubbed in

REMOVAL OF TEST SUBSTANCE
- Washing: deionized water
- Time after start of exposure: at the end of the daily exposure

TEST MATERIAL
- Amounts applied: depending on the dosage/ body weight/ day basis

Two positive control groups were employed:
Aspirin, 250 mg/kg bw per gavage and hexafluroacetone, 10 mg/kg bw.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, collars from gestation day 5 through gestation day 16

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1, in sufficient numbers to produce 120 pregnant females
- Verification of same strain and source of both sexes: yes, all animals were received from the same breeder
- Proof of pregnancy: vaginal plug referred to as day 0 of gestation

One male was not allowed to impregnate more than one female per treatment group.

Duration of treatment / exposure:

day 6 through day 15 of gestation

Frequency of treatment:

8 hours/day

Duration of test:

20 days

Dose / conc.:

75 mg/kg bw/day

Dose / conc.:

237 mg/kg bw/day

Dose / conc.:

750 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

Remarks:

positive control A: Hexafluoroacetone

Dose / conc.:

250 mg/kg bw/day

Remarks:

positive control B: Aspirin

No. of animals per sex per dose:

25

Control animals:

yes

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dosage regimen was selected based on the results of a preceding dose range-finding study (Becci et al., Fundam. Appl. Toxicol. 2, 1982).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on days 0, 6, 9, 12, 15 and 20 of gestation

FOOD CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and ovaries, gross necropsy

OTHER: dead fetuses

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Other: Number of dead and live fetuses, sex of each fetus

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one third of all fetuses and any fetus showing external abnormalities
- Skeletal examinations: Yes: all remaining fetuses
- Head examinations: No data

Statistics:

Incidences of occurrence were expressed as percent and comparisons between the negative control and test groups were made using either 95 % confidence intervals for proportions or by computations of exact probabilities.
- One-way ANOVA for fixed effects: continuous data
- Differences deemed significant p < 0.05
- Least significant difference test used to determine differences to negative control
- Unit of observation = dam or litter

Indices:

None

Historical control data:

None

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

dry skin patches (the severity of which increased with increasing dosage) on the dosing site and yellow-colored urine

Dermal irritation (if dermal study):

no effects observed

Description (incidence and severity):

only dry skin patches as discussed above

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw/day: marked decrease in body weight gain

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

no effects on number of implantations per dam

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw/day: percentage of implantation sites resorbed was significantly greater than the control; however, the percentage of dams with resorption sites was no different from the control

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw/day: significantly lowered number of live fetuses per dam

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

no effects on pregnancy rate

Dose descriptor:

NOAEL

Effect level:

237 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Dose descriptor:

LOAEL

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Effect level:

237 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

dead fetuses

early or late resorptions

Dose descriptor:

LOAEL

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

dead fetuses

early or late resorptions

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw/day: reduced fetal body weight of the surviving fetuses

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw/day: reduced number of live fetuses per litter

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw/day: skeletal alterations

Visceral malformations:

no effects observed

Description (incidence and severity):

no treatment-related effects on soft tissues (increased incidence of incomplete ossification of vertebrae, missing sternebrae, fused/split or extra ribs, incomplete closure of the skull, fused atlas and occipital bones of the skull, missing mandible and reduced hyoid)

Details on embryotoxic / teratogenic effects:

At 750 mg/kg bw/day, increased resorption rate, reduced number of live fetuses per litter, and reduced fetal body weight of the surviving fetuses were noted. The increased resorption rate was due to two dams with full litter resorptions and to three other dams with three to five resorptions per litter, which had live fetuses as well.
Visceral examination of fetuses revealed no treatment-related effects on soft tissues in fetuses for NMP-treated dams at any dose. Skeletal alterations were observed in the fetuses at 750 mg/kg bw/day only. These included increased incidence of incomplete ossification of vertebrae, missing sternebrae, fused/split or extra ribs, incomplete closure of the skull, fused atlas and occipital bones of the skull, missing mandible and reduced hyoid.
No adverse effects on the offspring were observed at 75 and 237 mg/kg bw/day. The fetuses from the two positive control groups exhibited visceral and skeletal malformations.

Dose descriptor:

NOAEL

Effect level:

237 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Dose descriptor:

LOAEL

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: rib

skeletal: vertebra

Description (incidence and severity):

While possibly being indicators of teratogenic potential, it should be noted that these abnormalities occurred only at a level of N-Methylpyrrolidone which could be considered maternally toxic .

Developmental effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

MATERNAL DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	75	237	750
Mated females [n]	25	25	25	25
Pregnant females [n]	24	22	23	24
Mortality of dams [n]	0	0	0	0
Clinical symptoms	none	mild^#	mild^#	mild^#
Body weight gain (days 6-15) [g]	38.9	38.1	42.3	28.1*
^#: colored urine and dry patches of skin (increased severity with increasing dose levels), *: p < 0.05

CAESARIAN SECTION / FETAL EXAMINATION
Endpoint	Dose Group [mg/kg bw/day]
	0	75	237	750
Mated females [n]	25	25	25	25
Pregnant females [n]	24	22	23	24
Aborted [n]	0	0	0	0
Implantation sites / dam, mean [n]	11.8	12.1	12.2	11.0
Live fetuses / dam, mean [n]	11.2	11.9	12.0	9.3*
Total resorptions [n]	15	6	4	41*
Dead fetuses [n]	0	0	0	0
Fetal weights, mean [g]	3.45	3.49	3.54	2.83*
*: p < 0.05

Conclusions:

NMP caused clear maternal toxicity (marked decrease in body weight gain) at 750 mg/kg bw/day. Fetotoxic effects consisted of fewer live fetuses, increased resorption rate, reduced fetal weight and indications of retarded skeletal development as well as an increased appearance of skeletal malformations (e.g., fused, surplus or cleft ribs, fusion of skull bones) at the high dose level occurred only at a dose level of marked maternal toxicity.
The NOAEL for maternal toxicity, developmental toxcity and teratogenicity was each 237 mg/kg bw/day.

Executive summary:

Prenatal developmental toxicity of NMP after dermal application of 0, 75, 237 and 750 mg/kg bw/day was investigated in groups of 25 Sprague Dawley rats. The test compound was applied unchanged under open conditions to an area of 25 cm² for eight hours daily from gestation day 6 through day 15. The dams were fitted with collars to prevent ingestion of the test compound.

Reduced body weight gain by 28 % was noted at 750 mg/kg bw/day. NMP caused clear maternal toxicity (marked decrease in body weight gain) at 750 mg/kg bw/day. The dams treated showed topical signs of irritation in a dose-dependently increased severity as well as colored urine as an indication of systemic test substance availability. Fetotoxic effects consisted of fewer live fetuses, increased resorption rate, reduced fetal weight and indications of retarded skeletal development as well as an increased appearance of skeletal malformations (e.g., fused, surplus or cleft ribs, fusion of skull bones) at the high dose level.

Thus, embryo-/fetotoxicity including malformations occurred only at a dose level of marked maternal toxicity. The NOAEL for maternal toxicity, developmental toxcity and teratogenicity was each 237 mg/kg bw/day.

Reference 8

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-04-03 to 1991-05-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

12 May 1981

Deviations:

no

Principles of method if other than guideline:

Method: Directive 87/302/EEC, part B, p. 24, 18 November 1987

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, from continuous production, tank No. 53
- Analytical purity: 99.8 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: under N2, to be protected from air
- Stability under test conditions: stability ensured by substance supplier

Species:

rabbit

Strain:

Himalayan

Remarks:

Chbb:HM

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Rationale for animals used: extensive experience is available on Himalayan rabbits and this strain had been proved to be sensitive to substances with a teratogenic potential
- Source: Dr. K. Thomae GmbH, Biberach, Germany
- Age at study initiation: 20 - 22 weeks old
- Weight at study initiation: approximately 2,415 g (mean)
- Housing: singly, stainless steel wire-mesh cages type K 300/8 (Becker & Co., Castrop-Rauxel, Germany)
- Diet: commercial diet (Kliba rabbit laboratory diet, 24-341-4, 10 mm pellets, Klingenthalmühle AG, Kaiseraugst, Switzerland), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours

IN-LIFE DATES: From: 11 March 1991, To: 15 May 1991

Route of administration:

dermal

Vehicle:

water

Remarks:

doubly distilled water

Details on exposure:

TEST SITE
- Area of exposure: 18 × 11.5 cm patch
- Type of wrap if used: stretchable bandage (Fixomull, Beiersdorf AG, Hamburg, FRG), semiocclusive dressing
- intact shaven dorsal skin, not more specified

REMOVAL OF TEST SUBSTANCE
- Washing: luke-warm water
- Time after start of exposure: on post insemination day 19 (end of exposure/organogensis)

TEST MATERIAL
- Amounts applied: different volumes of test substance solution. The calculation of the volume applied was based on the individual body weight determined at the beginning of the application period (day 7 p.i.).
- Constant volume or concentration used: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in doubly distilled water were carried out before the beginning of the study in the analytical laboratories of BASF Aktiengesellschaft. Furthermore, samples of the preparation of the test substance in doubly distilled water (40 g NMP/100 mL) were sent to the analytical laboratories of BASF Aktiengesellschaft twice during the study period for verification of the concentration. The test substance solutions were analyzed by gas chromatography.

Details on mating procedure:

- Impregnation procedure: artificial insemination, after the acclimation period
- Verification of same strain and source of both sexes: yes, the pooled ejaculate samples used were derived from male Himalayan rabbits of the same breed.
The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 post insemination (p.i.)

Duration of treatment / exposure:

day 7 through 19 of gestation

Frequency of treatment:

6 hours/day

Duration of test:

29 days

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

nominal in water

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

nominal in water

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

nominal in water

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on previous range-finding studies.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once per day

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed on days 0, 2, 4, 7, 9, 11, 14, 16, 19, 21, 23, 25 and 29 p.i.

FOOD CONSUMPTION: Yes
- Time schedule: Maternal food consumption was determined daily.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: ovaries and uterus, gross necropsy

OTHER:
The site of application was examined twice daily, once prior to and once upon termination of each 6-hour exposure period (days 7 - 19 p.i.)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: dead fetuses (hypoxemic fetuses whlch did not breathe spontaneously after the uterus had been opened)

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes, if heads revealed severe findings

Statistics:

Dunnett's test: food consumption, body weight, body weight change, corrected body weight gain, uterus weight, fetal and placental weights, corpora lutea, implantations, pre- and post-implantation losses, resorptions, live fetuses
Fisher's exact test: conception rate, mortality of dams, fetal findings

Indices:

Conception rate, pre- and post-implantation loss

Historical control data:

Historical control data from the breeder (external, skeletal and visceral defects) from around 2900 litters and 19000 fetuses were included in the report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/day (gestation days 10 - 16): reddish-brown discoloration of the urine
1000 mg/kg bw/day (gestation days 9 - 21): reddish-brown discoloration of the urine
- indicating NMP uptake through the skin

Dermal irritation (if dermal study):

no effects observed

Description (incidence and severity):

- no signs of irritation

Mortality:

no mortality observed

Description (incidence):

None of the does died during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no adverse effects on maternal body weights or body weight gains.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on feed consumption.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

on gestation day 29: no effects on uterine weight observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

on gestation day 29: no treatment-related maternal gross necropsy findings

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- no treatment-related or statistically significant differences among groups for pre- or post-implantation loss

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

- no treatment-related or statistically significant differences among groups forresorptions

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

All does were pregnant except for one at 100 mg/kg bw/day and three at 300 mg/kg bw/day.

Details on maternal toxic effects:

Pregnancy rate was 100 % at 0 and 1000 mg/kg bw/day, 93 % (14/15) at 100 mg/kg bw/day and 80 % (12/15) at 300 mg/kg bw/day.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

Remarks on result:

other: no adverse effect observed

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

- fetal body weights were equivalent across groups
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- no treatment-related or statistically significant differences for dead or live fetuses

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- no treatment-related or statistically significant differences for sex ratio per litter

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

- no effects on the number of abnormalities observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

- no effects on the number of abnormalities observed
- skeletal variations observed in all groups (involved the skull bones, ribs, vertebral column, and sternebrae)
- most skeletal variations did not exhibit a dose-response pattern of incidence
- number of fetuses with accessory 13th rib(s) was statistically significantly increased at 1000 mg/kg bw

Visceral malformations:

no effects observed

Description (incidence and severity):

- no effects on the number of abnormalities observed

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

Description (incidence and severity):

A slight increase in a common variation (supernumerary 13th ribs) in this strain of rabbits at the top dose group was observed.

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: vertebra

other: skeletal: talus ossification

Description (incidence and severity):

- treatment-relevant variations were only biologically relevant for the localisations mentioned

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Test substance analysis: The stability of the test substance formulation was established over a period of 7 days at room temperature. The correctness of the prepared concentrations was proven.

Table 1: Maternal parameters from pregnant Sprague-Dawley rats given NMP in the study.

MATERNAL DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	100	300	1000
Inseminated does on study [n]	15	15	15	15
Pregnant females [n]	15	14	12	15
Mortality of does [n]	0	0	0	0
Clinical symptoms	none	none	minimal^#	minimal^#
Food consumption (days 7-19) [g]	95.3	89.0*	96.8	91.4
Body weight gain (days 7-19) [g]	40.1	11.0	73.0	43.4
^#: reddish brown discoloration in urine, *: p < 0.05

Table 2: Gestational parameters from pregnant Srague-Dawley rats and incidence of malformations in foetuses of Sprague-Dawley rats given NMP.

CAESARIAN SECTION / FETAL EXAMINATION
Endpoint	Dose Group [mg/kg bw/day]
	0	100	300	1000
Inseminated does [n]	15	15	15	15
Pregnant does [n]	15	14	12	15
Conception rate [%]	100	93	80	100
Aborted [n]	0	0	0	0
Premature births [n]	0	0	0	0
Mortality of does [n]	0	0	0	0
Gravid uterus weight, mean [g]	343.1	319.7	374.0	383.5
Corpora lutea / doe, mean [n]	7.9	7.6	7.9	8.7
Implantation sites / doe, mean [n]	7.6	6.8	7.4	8.4
Live fetuses / doe, mean [n]	6.5	6.2	6.9	7.8
Total resorptions /doe, mean [n]	1.1	1.0	0.5	0.6
Dead fetuses / doe [n]	0	0	0	0
Placental weight, mean [g]	4.3	4.6	4.6	4.0
Fetal weights, males, mean [g]	39.2	41.9	41.1	36.9
Fetal weights, females, mean [g]	39.4	40.6	39.6	37.2
Accessory 13th rib(s) [fetal incidence, %]	1.0	1.2	6.0	15**
Total skeletal variations [fetal incidence, %]	18	15	19	29
All classified fetal observations
Total malformations [fetal incidence, %]	2.1	3.70	0	2.6
Total variations[ fetal incidence, %]	34	38	41	62**
Total retardations [fetal incidence, %]	57	54	61	68
**: p < 0.01

Conclusions:

The dermal application of aqueous NMP solutions to pregnant female Himalayan rabbits during organogenesis elicited no signs of maternal toxicity up to and including a dose level of 1,000 mg/kg body weight/day. The only substance­induced effect was a reddish-brown discoloration of the urine of the 300 and 1,000 mg/kg dams.

The only sign of developmental toxicity was the increased number of fetuses with accessory 13th rib(s) of dams exposed to 1,000 mg/kg. This effect is assessed as a non-specific embryotoxic effect.
No signs of embryo-/fetotoxicity were noted in the 100 or 300 mg/kg groups.

There were no indications of any teratogenic effect up to and including the highest dose level.

Executive summary:

In a developmental toxicity study, Himalayan rabbits (15 per group) were dermally exposed to a 40 % aqueous solution of NMP under a semiocclusive dressing for 6 hours daily (BASF SE, 1993).

Doses of 0, 100, 300 or 1000 mg/kg bw/day were applied from days 7 – 19 of pregnancy.

No adverse effects clinically or on food intake or body weight were observed in the dams although yellow urine indicated that absorption had occurred. There was no increase in malformations in the treated animals. The only treatment-related effect was a slight increase in a common variation (supernumerary 13th rib(s)) in this strain of rabbits at the high dose group only.

The NOAELs for maternal toxicity and teratogenicity were 1000 mg/kg bw/day, for developmental toxicity 300 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

360 mg/m³

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

237 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Oral route:

In a prenatal developmental toxicity study, pregnant Sprague-Dawley rats were treated by oral gavage with aqueous NMP solutions of 0, 125, 250, 500 or 750 mg/kg bw/day during gestational days 6 through 20 (Saillenfait, et al., 2002).

Significant decrements in maternal body weight gain and food consumption between treatment days 6 – 21 were observed at doses of 500 mg/kg bw/day onwards (Saillenfait, et al., 2002). If one considers biologically relevant effects, the maternal body weight gain was reduced by 9 % at 250 mg/kg bw/day, a reduction comparable to the statistically significant reduction in fetal body weight observed at the same dose. In addition, the net weight change was reduced by 11, 26 and 26 % at 250, 500 and 750 mg/kg bw/day, respectively.

Post implantation losses and the number of resorptions were increased at 500 mg/kg bw/day, showing a steep dose-response relationship. The rate of fetal malformations (external, skeletal, soft tissue) was increased at 500 mg/kg bw/day onwards. The principal types of malformations consisted of external (anasarca, anal atresia), soft tissue (persistent truncus arteriosus) and skeletal findings (fusion or absence of cervical arches were most prominent). The isolated occurrence of anal atresia and absent tail observed in one of 304 fetuses in one out of 24 litters at 250 mg/kg bw/day should be regarded as an incidental finding. It is not considered compound related as this type of malformation also occurs spontaneously with low frequencies in this rat strain.

Further findings of developmental toxicity were reduced fetal weights at 250 mg/kg bw/day onwards, delayed ossification of skull bones and sternebrae and an increase in skeletal variations at 500 mg/kg bw/day onwards. There was also a very low proportion of live fetuses and an increase in the rate of soft tissue variations at 750 mg/kg bw/day.

The NOAEL for maternal toxicity and developmental toxicity was 125 mg/kg bw/day considering biologically relevant impairments in maternal and fetal body weight. The NOAEL for malformations was 250 mg/kg bw/day.

It is noteworthy to mention that this assessment in respect to maternal toxicity is in contrast to the assessment of the authors that "(...) the NOAEL for maternal and developmental toxicity was 250 and 125 mg/kg/day, respectively".

In a further developmental toxicity study, groups of 20 inseminated New Zealand White rabbits were administered by oral gavage dose levels of 0, 55, 175 and 540 mg/kg bw/day of an aqueous NMP solution on gestation day 6 through 18 (IRDC, 1991).

Maternal toxicity was present at 175 and 540 mg/kg bw/day, expressed as significantly reduced body weight gain at both doses (marked at 540 mg/kg bw/day), reduced feed consumption at 540 mg/kg bw/day, and one abortion at 540 mg/kg bw/day. At 175 mg/kg bw/day, maternal body weight gain was transiently reduced on gestation days 6-12. Developmental toxicity was observed at 540 mg/kg bw/day in form of increased post-implantation loss, due to increased early and late resorptions, reduced live litter size, and reduced mean uterine weight.

Malformations observed at 540 mg/kg bw/day were related to the cardiovascular system and the skeleton (skull bones). No embryo-/fetotoxic effects or malformations were noted at lower dose levels.

As the maternal effects were observed at higher concentrations and appear to follow a dose response relationship, the effects seen at 175 mg/kg bw/d were taken into account in setting the NOAEL for maternal toxicity of 175 mg/kg bw/day. The NOAEL of 175 mg/kg bw/day was assumed for both developmental toxicity and malformations.

Inhalative route:

Pregnant Charles River CD Sprague-Dawley rats were exposed to N-methyl-2-pyrrolidone (NMP) at atmospheric concentrations of 0.1 and 0.36 mg/liter for 6 hr/day on pregnancy days 6 through 15 of gestation (Lee et al., 1987). Except for sporadic lethargy and irregular respiration in several rats the first 3 days of exposure, there were neither abnormal clinical signs nor pathological lesions in the maternal rats. Exposure did not affect either the outcome of pregnancy or embryonal growth rate. No abnormal development was detected in the vital organs and skeletons of the fetuses.

The NOAEC observed in this study is 0.36 mg/l which is equivalent to 360 mg/m3 (87 ppm).

 

The developmental toxicity of inhaled N-methyl-2-pyrrolidone (NMP) was studied in Sprague-Dawley rats. Pregnant rats were exposed whole body to NMP vapours at concentrations of 0, 30, 60 and 120 ppm, 6 h/day, on gestational days (GD) 6 through 20 (Saillenfait et al., 2003).

Maternal body weight gain was significantly decreased at 60 and 120 ppm on GD 6-13 and maternal food consumption was reduced at 120 ppm on GD 13-21 . No significant difference in the gestational weight change corrected for the weight of the gravid uterus was observed, whatever NMP concentration. There were no adverse effects on embryo/fetal viability or evidence of teratogenicity at any concentration tested. Fetal toxicity indicated by reduced fetal weight was observed at 120 ppm.

Thus, the no-observed-adverse-effect concentration (NOAEC) for maternal and developmental toxicity was 30 and 60 ppm, respectively.

 

As part of a reproduction toxicity study (Solomon et al., Drug. Chem. Toxicol. 18, 1995) 10 male and 20 female Sprague-Dawley rats were exposed to 0 or 116 ppm (478 mg/m3) NMP vapor starting at 34 days of age for 6h/d. For the male rats, exposure continued until day 134 of age, the end of the mating period. The females were exposed to gestation day 20 (day 140 -144). Body weights of the dams were recorded on gestation day 1 and 21, and clinical signs were monitored daily before, during and after exposure. After sacrifice, dams were examined for gross pathological changes, livers were weighed, corpora lutea were counted and resorptions were determined by staining. The fetuses were sexed and subjected to external, visceral, and skeletal examinations.

The indices of reproductive performance for the NMP-exposed rats did not differ significantly from those obtained for the control rats. Rats exposed to 116 ppm had a detectable decrease in response to sound. No other signs of NMP-related toxicity were detected among the parental rats.

An exposure-related slight decrease in pup weight was detected at 116 ppm.

The LOAEC for developmental toxicity was 116 ppm.

It has to be noted that the NOAEC for developmental toxicity based on a decrease in pup weight in the F1 offspring was reported as 206 mg/m3 (Solomon et al., Drug. Chem. Toxicol. 18, 1995). The NOAEC for maternal toxicity was reported in the study as 206 mg/m3 (51 ppm) (Solomon et al., Drug. Chem. Toxicol. 18, 1995). Due to the limited effect of reduced sensitivity to sound in female rats before mating, this NOAEC is considered as a systemic NOAEC.

 

Groups of 15 inseminated Himalayan rabbits were inhalation exposed to NMP concentrations of 0, 0.2, 0.5 and 1.0 mg/L (0, 49, 122, 243 ppm, vapor and vapor-aerosol mixture) for 6 hours daily on gestation day 7 through 19 (BASF SE, 1993).

The animals were treated in whole body inhalation chambers, sitting in specially shielded cages to avoid contamination of body surface (head-nose exposure).

No mortality occurred and no signs of maternal toxicity were noted in the examined parameters (clinical findings, body weight, body weight gain, corrected body weight, gross pathology) at any concentration.

However, a range-finding study (BASF SE, 21R0544/90049, 1991), which examined a wider range of parameters, showed maternal toxicity at 1.0 and 2.0 mg/L in form of increased liver weights and impaired clinical chemistry parameters. This concentration showed also slight fetotoxicity due to an increased incidence of supernumerary 13th ribs as a further indication of non-specific maternal stress and could be an evidence that a minimal toxic dose had been achieved.

The NOAEC for maternal toxicity and for developmental toxicity was 0.5 mg/L (122 ppm).

 

The range-finding study, which examined a wider range of parameters compared to the main study (BASF SE, 1993) showed maternal toxicity at 1000 (243 ppm) and 2000 (486 ppm) mg/m3 with increased liver weights and impaired clinical chemistry parameters. This concentration showed also slight fetotoxicity due to an increased incidence of supernumerary 13th rib(s) as a further indication of nonspecific maternal stress and could be evidence that a minimal toxic dose had been achieved.

As a combined approach, a NOAEC for maternal toxicity and for developmental toxicity of 500 mg/m3 (122 ppm) is suggested.

 

In a developmental toxicity study comparable to the OECD 414, pregnant female Wistar rats were exposed to clean air or 165 ppm NMP vapor from day 4 through day 20 of gestation (Hass et al., 1995).

No clinical signs of toxicity, no effects on mean food consumption or mean body weight gains (including body weight corrected for uterus weight) were observed in the dams. No significant differences concerning the number of resorptions or live fetuses per dam were recorded. There was a statistically significant increase in pre-implantation loss comparing 20/23 exposed to 11/20 non-exposed dams. The overall percentage of pre-implantation loss in exposed dams was 20.5+/-19.9% versus 13.4+/-16.4% in the control. However, no statistically significant or biologically relevant difference was observed in the post-implantation loss percentage (control: 15.6+/-15.1%, 165 ppm: 18.1+/-19.3%).

No biologically relevant differences in mean fetal body weights between treated and untreated rats were observed although reductions in fetal body weight per litter at 165 ppm were statistically significant. The profile of malformations observed at external, soft tissue and skeletal examination was similar between exposed and non-exposed rats. Delayed ossification of the skull bones, cervical vertebrae, and metatarsal and phalangeal bones was found at 165 ppm. For cervical vertebrae 4-7 and the phalangeal bones, the differences were statistically significant.

The LOAEC for maternal as well as developmental toxicity was 116 ppm (comparable to 618 mg/m3) based on the delayed physical development and the 6 -7% reduction of foetal body weight.

 

Pregnant rats (Mol: WIST) were exposed to 150 ppm N-methylpyrrolidone for 6 hours per day on gestation days 7-20. The dose level was selected so as not to induce maternal toxicity or decrease viability of the offspring (Hass et al., 1994). In the preweaning period, the exposed offspring had a lower body weight and their physical development was delayed. Neurobehavioral evaluation of the male pups revealed no effects on basal functions of the central nervous system. The animals appeared normal in motor function (rotarod), activity level (open field), and performance in learning tasks with a low grade of complexity were similar in the two groups. However, in more difficult tasks such as the reversal procedure in Morris water maze and operant delayed spatial alternation (Skinner boxes), performance was impaired in exposed offspring.

 

Dermal route:

Prenatal developmental toxicity of NMP after dermal application of 0, 75, 237 and 750 mg/kg bw/day was investigated in groups of 25 Sprague Dawley rats. The test compound was applied unchanged under open conditions to an area of 25 cm² for eight hours daily from gestation day 6 through day 15. The dams were fitted with collars to prevent ingestion of the test compound (GAF Corp. 1979).

Reduced body weight gain by 28 % was noted at 750 mg/kg bw/day. NMP caused clear maternal toxicity (marked decrease in body weight gain) at 750 mg/kg bw/day. The dams treated showed topical signs of irritation in a dose-dependently increased severity as well as colored urine as an indication of systemic test substance availability. Fetotoxic effects consisted of fewer live fetuses, increased resorption rate, reduced fetal weight and indications of retarded skeletal development as well as an increased appearance of skeletal malformations (e.g., fused, surplus or cleft ribs, fusion of skull bones) at the high dose level.

Thus, embryo-/fetotoxicity including malformations occurred only at a dose level of marked maternal toxicity. The NOAEL for maternal toxicity, developmental toxcity and teratogenicity was each 237 mg/kg bw/day.

 

In a further developmental toxicity study , Himalayan rabbits (15 per group) were dermally exposed to a 40 % aqueous solution of NMP under a semiocclusive dressing for 6 hours daily.

Doses of 0, 100, 300 or 1000 mg/kg bw/day were applied from days 7 – 19 of pregnancy (BASF SE, 1993).

No adverse effects clinically or on food intake or body weight were observed in the dams although yellow urine indicated that absorption had occurred. There was no increase in malformations in the treated animals. The only treatment-related effect was a slight increase in a common variation (supernumerary 13th rib(s)) in this strain of rabbits at the high dose group only.

The NOAELs for maternal toxicity and teratogenicity were 1000 mg/kg bw/day, for developmental toxicity 300 mg/kg bw/day.

 

In a dose-range finding study performed before a teratotoxicity study (FDRL, 1979), 5 pregnant female Sprague-Dawley rats per dose were administered NMP dermally. NMP was applied on days 6 through 15 of gestation at an area of approximately 25 cm2. The dose levels were 500, 1100 and 2500 mg/kg bw/day and the substance was applied daily. Hexafluoroacetone was used as a postivie control in a 25% solution at a level of 5 mg/kg bw/day.

Maternal animals in all NMP-exposed groups exhibited bright yellow urine and dry patches of skin on the dosing site. At 2500 mg NMP/kg bw, all pregnant four treated females died. At 1100 mg/kg bw NMP was embryolethal (65 of 66 implants were resorbed). The dams at this dose exhibited reduced weight gain partly due to the embryolethality. Dams at 500 mg NMP/kg bw exhibited no effect in body weights or weight gains. The one live fetus at 11000 mg NMP/kg bw was significantly smaller (2.07 g) than the negative control fetuses (mean 3.14 g). No external malformations or abnormalities were noted in the fetuses from NMP-treated dams.

In this limited study (dose-range finding) the NOAEL for both, maternal and developmental toxicity was 500 mg/kg bw/day. Based on the results of this study, the dose levels of NMP in the main teratotoxicity study were set to 75, 237 and 750 mg/kg bw/day.

Justification for classification or non-classification

Based on the key study results, NMP is not subject to classification and labelling for effects on fertility. Based on the outcomes of the developmental toxicity / teratogenicity key studies, NMP is legally classified in the EU (Annex VI of the Regulation (EC) No 1272/2008) as follows:

Repr. Cat 1B, H360D May damage the unborn child.