1-Butanone, 2-(dimethylamino)-2-[(4-methylphenyl)methyl]-1-[4-(4-morpholinyl)phenyl]-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The histopathological effects observed in the OECD 421 study were also observed in the subacute oral toxicity study.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx 11 weeks
- Weight at study initiation: (P) Males: 315 - 346 g; Females: 189-216 g
- Fasting period before study: no
- Housing: 5 animals/sex/cage (pre-mating, also males post mating), Females were caged together with males on a one-to-one-basis during mating and individually post mating.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 April 2013 To: 11 June 2013

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made
for specific gravity of the vehicle

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is not soluble in water.
- Concentration in vehicle: adjusted to dose
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum.
- Further matings after two unsuccessful attempts: no (not required in guideline)
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (0.5 mL) were taken using a pipette with the tip cut off (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice.

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

28 days (males)
42 - 52 days (females)

Frequency of treatment:

daily

Details on study schedule:

- Age at mating of the mated animals in the study: 13 weeks

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Range-finder study
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.
- Cage side observations included viability/morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, at least conducted approximately 1 to 2 hours after dosing.


BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

Parameters examined in male parental animals:
testis weight, epididymis weight, staging of spermatogenesis

Litter observations:

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

GROSS EXAMINATION OF DEAD PUPS:
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk. If possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals after 28 days.
- Maternal animals: All surviving animals at day 5-7 post partum
- Females which failed to deliver: Post-coitum Day 27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
- Females with total litter loss: within 24h after total litter loss

GROSS NECROPSY
- Gross necropsy consisted of external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Epididymides and testes were weighed.
Histopathology investigation was peformed on the ovaries of the females of Groups 1 and 4, the testes and epididymides of the males of all groups, the additional slides of the testes of the males of all groups to examine staging of spermatogenesis and all gross lesions of all animals (all dose groups).
For animals not mated or not pregnant, the following organs were examined histopathologically: cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.
For animals with total litter loss, the mammary gland area was investigated.

The numbers of former implantation sites and corpora lutea were recorded for all paired females

Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS: not investigated

Statistics:

If the variables could be assumed to follow a normal distribution, the Dunnett-test (manyto-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test was applied to frequency data.

Reproductive indices:

Mating index (%), fertility index (%), conception index (%), gestation index (%), duration of gestation

Offspring viability indices:

Percentage live males at first Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss, Viability index

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw day, treatment related clinical signs consisted of piloerection (six females), hunched posture (three females), and one female (no. 71) also showed lethargy and pale appearance.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight gain was noted for males at 60 and 200 mg/kg bw/day. Females at 200 mg/kg bw/day showed a body weight loss during the first week of treatment; this recovered during the remainder of the study period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight gain was noted for males at 60 and 200 mg/kg bw/day. Females at 200 mg/kg bw/day showed a body weight loss during the first week of treatment; this recovered during the remainder of the study period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in testes and epididymides of males treated at 200 mg/kg bw/day and consisted of:
- Intraluminal cell debris of the epididymides in 8/10 males (up to moderate degree).
- Oligospermia of the epididymides in 4/10 males (up to slight degree).
- Germ cell exfoliating into the lumen of seminiferous tubules of the testes (without degeneration) in 9/10 males (up to moderate degree).

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Findings of note were recorded in the kidneys and thymus of females treated at 200 mg/kg bw/day and consisted of:
- Glomerular and tubular necrosis (marked) of the kidneys in 1/10 females, noted at necropsy as many reddish foci (both sides).
- Lymhoid atrophy (moderate) of the thymus in 2/10 females, noted at necropsy as gelatinous or reduced size. These two females also showed total litter loss.

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

Dose descriptor:

NOEL

Remarks:

reproductive performance (fertility)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive indeces were not affected.

Critical effects observed:

not specified

Lowest effective dose / conc.:

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

System:

other: Detailed investigations on organ toxicity are not part of a screening study for reproductive toxicity.

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

200 mg/kg bw - This consisted of an increased number of dead pups at first litter check and postnatal loss, and a decreased number of living pups at first litter check and viability index. These findings were mainly caused by two litters of which all pups died on Day 1 or 2 of lactation (all twenty pups that were found dead at first litter check and 7 out of 12 pups that died later). One of these dams showed marked glomerular and tubular necrosis of the kidneys which probably had an impact on the animal’s health and could have resulted in her total litter loss on Day 1 of lactation. This could be regarded as an indirect effect on pup development for this litter, however this was not the case for the other pup deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day body weights of the pups were reduced on Days 1 and 4 of lactation.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Dose descriptor:

NOEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Remarks on result:

other: observation period postnatal days 1-4

Critical effects observed:

not specified

Reproductive effects observed:

no

REPRODUCTION DATA SUMMARY

	control	20mg/kg bw	60 mg/kg bw	200 mg/kg bw
Females paired	10	10	10	10
Females mated	9	10	10	10
Pregnant females	8	10	9	10
Females with living pups on Day 1	8	10	9	9
Mating index (%) (Females mated / Females paired) * 100	90	100	100	100
Fertility index (%) (Pregnant females / Females paired) * 100	80	100	90	100
Conception index (%) (Pregnant females / Females mated) * 100	89	100	90	100
Gestation index (%) (Females with living pups on Day 1 / Pregnant females) * 100	100	100	100	90

BODY WEIGHTS (GRAM), males
		control	20mg/kg bw	60 mg/kg bw	200 mg/kg bw
PREMATING DAY1	MEAN	327	327	326	331
WEEK1	ST.DEV	8.1	12.0	5.8	7.6
	N	10	10	10	10
DAY8	MEAN	348	346	340	335*
WEEK2	ST.DEV	10.8	13.0	7.6	9.2
	N	10	10	10	10
MATINGPERIOD DAY1	MEAN	366	362	353	346**
WEEK1	ST.DEV	13.5	13.5	8.7	10.2
	N	10	10	10	10
DAY8	MEAN	375	374	362	353**
WEEK2	ST.DEV	15.9	15.0	8.1	12.2
	N	10	10	10	10
DAY15	MEAN	392	389	376	367**
WEEK3	ST.DEV	16.5	17.7	8.5	15.7
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

BODY WEIGHTS (GRAM), females
		control	20mg/kg bw	60 mg/kg bw	200 mg/kg bw
PREMATING DAY1	MEAN	201	204	202	204
WEEK1	ST.DEV	7.1	9.0	5.7	10.3
	N	10	10	10	10
DAY8	MEAN	206	203	204	196
WEEK2	ST.DEV	10.4	6.9	8.3	10.0
	N	10	10	10	10
MATINGPERIOD DAY1	MEAN	211	213	213	211
WEEK1	ST.DEV	7.4	8.1	9.5	7.4
	N	10	10	10	10
DAY8 WEEK2	MEANST.DEVN	231 --- 1		226 --- 1
DAY15 WEEK3	MEANST.DEVN	249 --- 1
DAY22 WEEK4	MEANST.DEVN	238 --- 1
DAY29 WEEK5	MEANST.DEVN	240 --- 1
POSTCOITUM DAY0	MEAN	213	213	214	207
	ST.DEV.	10.9	7.8	9.3	7.7
	N	8	10	9	10
DAY4	MEAN	225	227	226	220
	ST.DEV.	10.2	9.2	7.0	8.5
	N	8	10	9	10
DAY7	MEAN	233	235	236	229
	ST.DEV.	8.6	11.0	6.1	8.7
	N	8	10	9	10
DAY11	MEAN	248	248	249	240
	ST.DEV.	13.2	12.6	7.3	7.3
	N	8	10	9	10
DAY14	MEAN	261	263	261	254
	ST.DEV.	11.1	12.1	7.0	9.3
	N	8	10	9	10
DAY17	MEAN	283	285	284	274
	ST.DEV.	14.8	9.5	8.3	9.3
	N	8	10	9	10
DAY20	MEAN	319	317	315	305
	ST.DEV.	21.0	12.1	13.1	16.3
	N	8	10	9	10
LACTATION
DAY1	MEAN	243	247	244	230
	ST.DEV.	14.3	12.1	11.6	12.4
	N	8	10	9	10
DAY4	MEAN	261	260	257	252
	ST.DEV.	15.2	11.6	6.5	8.3
	N	8	10	9	8

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

CORPORA LUTEA AND IMPLANTATION SITES SUMMARY
		GROUP1	GROUP2	GROUP3	GROUP4
		CONTROL	20MG/KG	60MG/KG	200MG/KG
Corpora Lutea	MEAN	13.5	12.8	13.4	13.2
	ST.DEV	2.4	1.3	2.1	2.3
	N	8	10	9	10
Implantations	MEAN	12.5	11.2	12.2	12.6
	ST.DEV	1.7	2	1.6	2.5
	N	8	10	9	10

		CONTROL	20MG/KG	60MG/KG	200MG/KG
LITTERS
	Total	8	10	9	10
DURATION OF GESTATION
	MEAN (+)	21.5	21.3	21.7	21.4
	ST.DEV	0.5	0.5	0.7	0.7
	N	8	10	9	10
DEAD PUPS AT FIRST LITTER CHECK
	LITTERS AFFECTED (#)	0	2	1	2
	TOTAL	0	2	4	20
LIVING PUPS AT FIRST LITTER CHECK
	% OF MALES / FEMALES	48 / 52	59 / 41	45 / 55	46 / 54
	TOTAL	92	106	98	99
	MEAN	11.5	10.6	10.9	9.9
	ST.DEV	2	1.8	2.5	4.2
	N (litters)	8	10	9	10
POSTNATAL LOSS
	% OF LIVING PUPS	1.1	0	1	12.1
	LITTERS AFFECTED (#)	1	0	1	4
	TOTAL (#)	1	0	1	12 ##
	VIABILITY INDEX (#)	98.9	100	99	87.9 ##

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

+/++ Steel-test significant at 5% (+) or 1% (++) level

Viability index = (Number of alive pups before planned necropsy / Number of pups born alive) *100

BODY WEIGHTS OF PUPS (GRAM)
			GROUP1	GROUP2	GROUP3	GROUP4
	Sex		CONTROL	20MG/KG	60MG/KG	200MG/KG
		MEAN	6.3	6.2	6.3	5.4 *
	Male	ST.DEV	0.6	0.7	0.7	0.5
		N (litters)	8	10	9	9
		MEAN	6	6	6	5.1 *
Day 1	Female	ST.DEV	0.7	0.6	0.6	0.6
		N (litters)	8	10	9	9
		MEAN	6.2	6.1	6.1	5.3 *
	M/F	ST.DEV	0.6	0.6	0.7	0.6
		N (litters)	8	10	9	9
		MEAN	9.4	9.2	9.3	7.5 **
	Male	ST.DEV	1	1.2	1.3	0.9
		N (litters)	8	10	9	8
		MEAN	9	9	9	7.3 *
Day 4	Female	ST.DEV	1.2	1.2	1.3	1.1
		N (litters)	8	10	9	8
		MEAN	9.2	9.1	9.1	7.4 *
	M/F	ST.DEV	1.1	1.2	1.3	1
		N (litters)	8	10	9	8
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Conclusions:

The substance does not affect fertility. It affects pup development at maternally toxic doses.

Executive summary:

In this guideline (OECD 421) study conducted with GLP certification, the test material was found to provoke no reproductive toxicity in rats. The test was conducted at three dose levels (20, 60 and 200 mg/kg bw/day). The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females. No treatment related toxicity or treatment related reproductive toxicity was observed in the parental or offspring animals. The NOEL was set at the 60 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A screening study (OECD 421) is available.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No reproduction toxicity was observed up to the highest dose level tested (200 mg/kg bw/day ) in the screening study in rats (OECD 421, GLP). Slight histopathology findings did not result in effects on male fertility. Specifically, these effects were observed at 200 mg/kg bw and consisted of  slightly lower epididymis weights in males (approx. 20%), intraluminal cell debris in the epididymides (8 males, up to moderate degree),  intraluminal oligospermia in the epididymides (4 males, up to slight degree) and germ cell exfoliating into the lumen of seminiferous tubules in the testes (9 males, up to moderate degree). For details it is referred to the attached CLH report for reproductive toxicity of July 2016.

Short description of key information:  No impairment of fertility was observed in rats in a screening study with gavage dosing (OECD 421, GLP) at the highest dose level of 200 mg/kg bw.

Effects on developmental toxicity

Description of key information

Developmental toxicity in rats was observed at 200 mg/kg bw/day (BASF 2013). This consisted of an increased number of dead pups at first litter check and postnatal loss, and a decreased number of living pups at first litter check and viability index. These findings were mainly caused by two litters of which all pups died on Day 1 or 2 of lactation (all twenty pups that were found dead at first litter check and 7 out of 12 pups that died later). One of these dams showed marked glomerular and tubular necrosis of the kidneys which probably had an impact on the animal’s health and could have resulted in her total litter loss on Day 1 of lactation. This could be regarded as an indirect effect on pup development for this litter, however this was not the case for the other pup deaths. In addition, at 200 mg/kg bw/day body weights of the pups were reduced on Days 1 and 4 of lactation. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 421

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx 11 weeks
- Weight at study initiation: (P) Males: 315 - 346 g; Females: 189-216 g
- Fasting period before study: no
- Housing: 5 animals/sex/cage (pre-mating, also males post mating), Females were caged together with males on a one-to-one-basis during mating and individually post mating.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 April 2013 To: 11 June 2013

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made
for specific gravity of the vehicle

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is not soluble in water.
- Concentration in vehicle: adjusted to dose
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum.
- Further matings after two unsuccessful attempts: no (not required in guideline)
- After successful mating each pregnant female was caged individually

Duration of treatment / exposure:

28 days (males)
42 - 52 days (females)

Frequency of treatment:

daily

Duration of test:

28 days (males)
42 - 52 days (females)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

it is referrred to the robust study summary for fertility as this is the routine place in IUCLID for entering a OECD 421 study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.
- Cage side observations included viability/morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, at least conducted approximately 1 to 2 hours after dosing.


BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

Ovaries and uterine content:

not examined

Fetal examinations:

not examined

Statistics:

yes

Indices:

Percentage live males at first Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss, Viability index

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption (absolute and relative) was decreased during the first week of treatment and during lactation for females at 200 mg/kg bw/day.

Ophthalmological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Findings of note were recorded in the kidneys and thymus of females treated at 200 mg/kg bw/day and consisted of:
- Glomerular and tubular necrosis (marked) of the kidneys in 1/10 females, noted at necropsy as many reddish foci (both sides).
- Lymhoid atrophy (moderate) of the thymus in 2/10 females, noted at necropsy as gelatinous or reduced size. These two females also showed total litter loss.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day, there were possible treatment related macroscopic findings in kidneys and/or thymus for the two females that showed a total litter loss.
One female (no. 71) that had a total litter loss showed general pale discolouration, many reddish foci on the kidneys, gelatinous thymus, and the other (female no. 79) showed the thymus reduced in size.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

not specified

Description (incidence and severity):

One female (no. 71) had a total litter loss.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

no effects 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

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Details on maternal toxic effects:

Food consumption (absolute and relative) was decreased during the first week of treatment and during lactation for females at 200 mg/kg bw/day.

At 200 mg/kg bw/day, there were possible treatment related macroscopic findings in kidneys and/or thymus for the two females that showed a total litter loss.
One female (no. 71) that had a total litter loss showed general pale discolouration, many reddish foci on the kidneys, gelatinous thymus, and the other (female no. 79) showed the thymus reduced in size.

Findings of note were recorded in the kidneys and thymus of females treated at 200 mg/kg bw/day and consisted of:
- Glomerular and tubular necrosis (marked) of the kidneys in 1/10 females, noted at necropsy as many reddish foci (both sides).
- Lymhoid atrophy (moderate) of the thymus in 2/10 females, noted at necropsy as gelatinous or reduced size. These two females also showed total litter loss.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

histopathology: non-neoplastic

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, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 200 mg/kg bw/day body weights of the pups were reduced on Days 1 and 4 of lactation.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

Developmental toxicity was observed at 200 mg/kg bw/day. This consisted of an increased number of dead pups at first litter check and postnatal loss, and a decreased number of living pups at first litter check and viability index. These findings were mainly caused by two litters of which all pups died on Day 1 or 2 of lactation (all twenty pups that were found dead at first litter check and 7 out of 12 pups that died later). One of these dams showed marked glomerular and tubular necrosis of the kidneys which probably had an impact on the animal’s health and could have resulted in her total litter loss on Day 1 of lactation. This could be regarded as an indirect effect on pup development for this litter, however this was not the case for the other pup deaths.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

This consisted of an increased number of dead pups at first litter check and postnatal loss, and a decreased number of living pups at first litter check and viability index. These findings were mainly caused by two litters of which all pups died on Day 1 or 2 of lactation (all twenty pups that were found dead at first litter check and 7 out of 12 pups that died later). One of these dams showed marked glomerular and tubular necrosis of the kidneys which probably had an impact on the animal’s health and could have resulted in her total litter loss on Day 1 of lactation. This could be regarded as an indirect effect on pup development for this litter, however this was not the case for the other pup deaths.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Developmental toxicity was observed at 200 mg/kg bw/day.

Dose descriptor:

NOAEL

Effect level:

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

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.:

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

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

not specified

Summary tables are contained in the attachment to this endpoint study record.

It is also referrred to the robust study summary for fertility as this is the routine place in IUCLID for entering a OECD 421 study.

Conclusions:

The test material was determined to have a maternal toxicity NOEL of 60 mg/kg bw/day, and a fetal developmental NOAEL of 60.0 mg/kg bw/day (bodyweight) in the rat under the conditions of the test.

Executive summary:

In this guideline (OECD 421) study conducted with GLP certification, the test material was found to provoke developmental toxicity in rats. The test was conducted at three dose levels (20, 60 and 200 mg/kg bw/day). The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females. Treatment-related developmental toxicity was observed at 200 mg/kg bw/day. This consisted of an increased number of dead pups at first litter check and postnatal loss, and a decreased number of living pups at first litter check and viability index. The NOEL was set at the 60 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A full study for developmental toxicity/teratogenicity is not available. Results are based on a screening study (OECD 421).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A screening study (OECD 42, GLP) with dose levels of 200, 60 and 20 mg/kg bw is available. Dose levels were chosen based on the results of a 14 -day preliminary study. Findings observed were

200 mg/kg bw/day:

-       Two females were euthanized due to total litter loss. One of these female showed lethargy and a pale appearance on the day of total litter loss

-       Increased number of dead pups at first litter check (20 versus 0 in controls)

-       Increased number of postnatal loss (12 versus 1 in controls)

-       Lower pup weights at postnatal day 1 (approx. 15%) and 4 (approx. 20%)

-       Moderate lymphoid atrophy in the thymus in two females with total litter loss

-       Marked glomerular and tubular necrosis in the kidney in one of the aforementioned females

- Food consumption (absolute and relative) was decreased during the first week of treatment and during lactation for females at 200 mg/kg bw/day.

 

60 mg/kg bw/day and 20 mg/kg bw

-       No effects were observed.

For details it is referred to the attached CLH report for reproductive toxicity of July 2016.

Toxicity to reproduction: other studies

Additional information

A reliable evaluation with comments on the results concerning toxic effects on reproduction described in a 28-day oral toxicity (gavage) study in rats is available (Chahoud 2004). In the subacute toxicity study, the test item was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 0, 15, 50, 150 and 450 mg/kg body weight/day for a period of 28 days. In the control and high dose group additional animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

 

The test substance showed clear effects on testes and epididymes at only the dose level of 450 mg/kg:

- reduced organ weights of testes and epididymes compared to the control.

- reduced spermatogenesis in testes, in some cases associated with occurrence of spermatic giant cells and tubular atrophy.

- cellular debris and reduced amount of spermatozoa in epididymes.

However, signs of general toxicity were observed at the same dose level as well as after administration of 150 mg/kg. Considering the effects on body weight gain, the MTD was clearly exceeded in that study. It appears, therefore, that the male reproductive organs are not the most sensitive target tissues in this case. At the end of the 14 day treatment free recovery period after administration of 450 mg/kg, some general toxic effects as well as the effects on male reproductive organs could still be observed.

 

Effects on reproductive organs can not be considered as specific reproductive toxicity if signs of general toxicity are observed at the same dose level. Since signs of general toxicity were observed after administration of 150 mg as well as 450 mg/kg, the effects on male reproductive organs described above cannot be considered as a sign of reproductive toxicity of the test item. In general, effects on testes and epididymes of adult animals are considered to be reversible after a certain period of exposure-free time. Since spermatogenesis in the rat lasts several weeks, a recovery time of at least one spermatogenic cycle should be allowed.

Justification for classification or non-classification

It is referred to the attached CLH report for reproductive toxicity of July 2016.