Amines, N-(3-aminopropyl)-N'-C12-18-alkyltrimethylenedi-, reaction products with N-C12-18-alkyltrimethylenediamines and formic acid

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2010-05-04 to 2010-07-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

other: Draft report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: USA EPA Health Effects Test Guideline OPPTS 870.3650, July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Not assignable, UVCB

EC Number:

641-088-6

Cas Number:

1229648-98-9

Molecular formula:

No molecular formula

IUPAC Name:

Not assignable, UVCB

Details on test material:

- Name of test material (as cited in study report): Polyram L200 (without solvent)
- Physical state: dark brown liquid
- Analytical purity: 88.6%
- Composition of test material, percentage of components:
Condensation product of N-C12-C18-alkylpropane-1,3-diamine, N-(3-aminopropyl)-N'-C12-C18-alkylpropane-1,3-diamine and formic acid: 88.6%N-(C12-C18)alkyl dipropylenetriamide and N-(C12-C18)alkylpropylenediamide: 10%
Other: 1.4%
- Lot/batch No.: 81222503
- Purity test date: 29 January 2008
- Expiration date of the lot/batch: 29 January 2011
- Storage conditions of test material: at room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle, France.
- Strain and Sanitary status: Sprague-Dawley Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®)
- Age at study initiation: males: approximately 11 weeks old; females: approximately 10 weeks old
- Weight at study initiation: Males: 380 to 468 g; Females: 201 to 262 g
- Housing: the males and females were individually housed, except during pairing, in wire-mesh cages. Towards the end of the gestation period and with their litter during lactation, the females were housed in polycarbonate cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was mixed with the required quantity of vehicle in order to achieve the concentrations of 1, 3 and 9 mg/mL.
The dosage forms were prepared daily and were kept at room temperature in brown flasks. They were used within 4 hours of preparation.
The weighing print-outs were checked after every preparation and before delivery of the dosage forms to the animal room. The aspect of each dosage form was also recorded.

VEHICLE
- Justification for use and choice of vehicle :solubility
- Concentration in vehicle: 1, 3 and 9 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until mating occurred or 14 days had elapsed
- Proof of pregnancy: vaginal plug or sperm in vaginal smear . The day of confirmed mating was designed day 0 post-coitum.
- After successful mating each pregnant female was housed individually in wire-mesh cages.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

After many months of work trying to validate the analytical method for analysis of the dosage forms, it was concluded that the method could not be validated. As a deviation to GLP, chemical analysis of the dosage forms was therefore not conducted, however the weighing prints-out from the daily preparation of the dosage forms were checked and the aspect of the dosage forms were also checked daily. This exception is considered not to impact on the overall status of the study. A discussion with all the analytical researches performed will be provided with the final report of the study.

Duration of treatment / exposure:

- Males: 15 days before mating, during the mating period (up to 2 weeks) and until sacrifice (i.e. at least 4 weeks in total)
- Females: 15 days before mating, during the mating period (up to 2 weeks), during pregnancy, during lactation until day 5 post-partum inclusive.
- Recovery animals: same treatment schedule as the principal male groups but without mating and kept for an additional 4-week treatment-free period.
(day 1 = first day of treatment period)

Frequency of treatment:

Once daily.

Details on study schedule:

- No F1 parental animals: only one generation mated
- Age at mating of the mated animals in the study: approximately 13 to 15 weeks for males and 12 to 14 weeks for females

Doses / concentrations

Remarks:

Doses / Concentrations:
5, 15 and 45 mg/kg bw/day
Basis:
other: nominal

No. of animals per sex per dose:

- The low- and intermediate-dose groups were each composed of 10 males and 10 females.
- The high-dose group was composed of 15 males and 15 females.The first 10 animals/sex having been mated were sacrificed at the end of the treatment period ; the last 5 animals/sex not mated were dosed for 4 weeks and then retained for a 4-week treatment-free period.
- The control group was composed of 15 males and 15 females. As for the high-dose group, the first 10 animals/sex were mated and sacrificed at the end of the treatment period and the last 5 animals/sex were retained for a 4-week treatment-free period.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were selected on the basis of a 2-week toxicity study in the same species and strain in which dose-levels of 15, 45 or 150 mg/kg bw/day were administered. The dose-level of 150 mg/kg bw/day resulted in one premature sacrifice while at 45 mg/kg bw/day loud breathing, grey/white foci and thickening of forestomach wall were the only signs of toxicity (CIT 2010, study n°34507 TSR reported in chapter 7.5.1).

Positive control:

None.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day during the acclimation and treatment-free periods and at least twice a day during the treatment period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT: Yes
Time schedule for examinations:
- Males: on the first day of treatment (day 1), then once a week until sacrifice.
- Females: on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION: Yes
- Males: once a week, over a 7 day period, from the first day of treatment until sacrifice.
- Females: once a week, over a 7 day period, from the first day of treatment through gestation (days 0-7, 7-14 and 14-20 post-coitum intervals) and lactation (days 1-5 post-partum intervals) until sacrifice.
During the pairing period, food consumption was not recorded for males or females.

OTHER:
FUNCTIONAL OBSERVATION BATTERY (FOB) and LABORATORY INVESTIGATIONS were also performed (see section 7.5.1 : CIT 2010a / K1 KS/ Repeated dose toxicity: oral)

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the mating period, until the
females had mated.

Sperm parameters (parental animals):

Parameters examined in male parental generation (F0):
epididymis and testis weights

Litter observations:

STANDARDISATION OF LITTERS: Not applicable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups,
- postnatal mortality and cannibalized pups,
- presence of gross malformations,
- clinical signs,
- body weight (recorded on days 1 and 5 post-partum)

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
After overnight fasting, at least 14 hours, the following surviving F0 animals were deeply anesthetized by an intraperitoneal injection of sodium
pentobarbital and sacrificed by exsanguination.
- males: after the end of the mating period (after at least 4 weeks of treatment),
- females: on day 6 post-partum,
- 1 female which had not delivered by day 25 post-coitum was sacrificed on this day (without overnight fasting) by inhalation of carbon dioxide gas followed by cervical dislocation after body weight recording to check for a possible unnoticed delivery.

GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices,
the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the
neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
A careful examination of the stomach lining was performed due to the strong irritant / corrosive properties of the test item.
The numbers of corpora lutea and implantation sites were also recorded for females sacrificed on day 6 post-partum.
For the apparently non-pregnant female the presence of implantation scars on the uterus was checked using the ammonium sulphide staining
technique.

HISTOPATHOLOGY / ORGAN WEIGHTS
A microscopic examination was performed on:
- all the tissues listed in table 1 for the first five sacrificed-as-scheduled males and the first five females to deliver and be
sacrificed on day 6 post-partum of the control and high-dose groups (groups 1 and 4),
- all macroscopic lesions,
- selected tissues (forestomach, ileum and mesenteric lymph nodes) from animals sacrificed on completion of the treatment-free period and from
animals of the low- and intermediate-dose groups sacrificed at the end of the treatment period because of the results obtained at the end of the
treatment period.
The microscopic examination made special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

The body weight of each animal was recorded before sacrifice at the end of the treatment or treatment-free period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Postmortem examinations (offspring):

SACRIFICE
Surviving pups were sacrificed on day 5 post-partum, without fasting, by an intraperitoneal injection of sodium pentobarbital

GROSS NECROPSY
Pups found dead and pups sacrificed on day 5 post-partum were carefully examined externally for gross external abnormalities and a macroscopic
examination was performed.

Statistics:

- Body weights and reproductive data
Mean values were compared by one-way variance analysis and Dunnett test (mean values being considered as normally distributed, variances being considered as homogeneous).
Percentage values were compared by Fisher exact probability test.

- Organ weights
PathData software (version 6.2b5) was used for the statistical analysis of organ weight data (level of significance: 0.05 or 0.01)

Reproductive indices:

Pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

Post-implantation loss:
Number of implantation sites - Number of live concepti
_____________________________________________ x 100
Number of implantations

Mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

Fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

Gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females

Live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

Offspring viability indices:

Viability index on day 5 post-partum:
Number of surviving pups on day 5 post-partum
_______________________________________ x 100
Number of live born pups

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

clinical signs only

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

There were no unscheduled deaths during the study.

Hypersalivation was observed in males generally from weeks 2 to 4 of treatment at 45 mg/kg bw/day and in weeks 2 and 3 of treatment at 15 mg/kg bw/day, although in some males treated at 45 mg/kg bw/day it was observed as early as day 1 and sometimes lasted sporadically until week 6 of treatment. In females, hypersalivation was observed in the second week of the pre mating period and continued in most females throughout gestation and the beginning of the lactation period or until the end of the treatment period in females retained for the treatment-free period.

Loud breathing was observed in five males treated at 45 mg/kg/day between weeks 2 and 6 (from 2 days to 25 days) and in one male during the last 2 weeks of the treatment-free period. This last male also had round back and piloerection at the end of the treatment-free period. Six females treated at 45 mg/kg bw/dayalso had loud breathing; at the end of week 2 or week 4 for the two females which were mated, in weeks 2 to 5 in the females which were retained for the treatment-free period and towards the end of lactation period for two other females.

Half-closed eyes were observed in three males treated at 45 mg/kg bw/day for 2 to 4 days during weeks 2 or 4. Hypoactivity, half closed eyes, round back and piloerection were observed in up to five females of the high-dose group at the end of week 2 or the end of week 4 for females which were mated or around weeks 3, 4 and 5 for females which were retained for the treatment-free period. One of these females continued to have piloerection for the first week of gestation and another female, with no previous clinical signs other than hypersalivation, had hypoactivity and half-closed eyes for 2 days mid-gestation.
(see table 1 in remarks on results).


BODY WEIGHT AND WEIGHT GAIN (PARENTAL ANIMALS)

Males treated at 45 mg/kg bw/day gained statistically significantly less body weight during the first week of treatment when compared with the controls. Out of the 15 males, 7 males actually lost weight during this period (between -2 g and -19 g). Body weight gains were similar to those of the controls for the rest of the treatment period therefore the body weight deficit was not recouped and final mean body weight was 6% lower than that of the controls. Mean body weight remained lower throughout the treatment-free period because mean body weight gains were only similar to those of the controls and not higher, and therefore the mean body weight at the end of the treatment-free period was 10% lower than that of the controls.

Females treated at 45 mg/kg bw/day had mean body weight gains which were comparable with the controls during the treatment period but which were statistically significantly higher over the treatment-free period.

Mean body weights and mean body weight gains were comparable with the controls, or higher, in the male and female groups treated at 5 or 15 mg/kg bw/day.
(see table 2 in remarks on results).


FOOD CONSUMPTION (PARENTAL ANIMALS)

Correlating with the low body weight gain, during the first week of dosing males treated at 45 mg/kg bw/day had statistically significantly lower mean food consumption when compared with the controls (-22%, p<0.001). Food consumption was similar to that of the controls during the rest of the treatment period but was slightly lower during the treatment-free period. Females treated at the same dose level had slightly, but statistically significantly, lower mean food consumption during the first week of treatment (-11%, p<0.05). Mean food consumption remained slightly lower during the rest of the treatment period and during the gestation period but was comparable with that of the controls during the treatment-free and lactation periods.
The male and female groups treated at 5 or 15 mg/kg bw/day had comparable or slightly greater mean food consumption when compared with the
controls.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

One female treated at 45 mg/kg bw/day stayed in diestrus for 10 days but mated on the last day of pairing and was pregnant so it was considered that this abnormal estrous cycle was spontaneous in origin. No other females showed abnormal estrous cycling.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

- Mating (see table 3 in remarks on results)
All males and females mated and although the mean number of days taken to mate was longer in the test item-treated groups than in the control group, the values were all within normal biological range.
- Fertility (see table 4 in remarks on results)
One female treated at 45 mg/kg bw/day was not pregnant. There were no macroscopic changes at post-mortem examination. One non-pregnant female is considered to be within normal biological range.
- Delivery (see table 5 in remarks on results)
The mean duration of gestation was comparable with the controls and within normal biological range for all groups. The mean number of corpora lutea and the mean number of implantations were both comparable with the controls for all groups. The mean number of pups was moderately lower at 5 mg/kg bw/day and the post-implantation loss was higher. These effects were limited to the low-dose group and in no parameter has any evidence of an inverse-dose relationship been observed. Therefore, the lower number of pups was considered to be unrelated to treatment with the test item.


ORGAN WEIGHTS (PARENTAL ANIMALS)

In the animals sacrificed at the end of the treatment period, there were no changes in the mean organ weights which were considered to be associated with administration of the test item. The mean absolute and relative thymus weights were lower in both males and females given 45 mg/kg bw/day and in females given 5 or 15 mg/kg bw/day. As these values were not statistically significant, were poorly dose-related (females), were found in isolated animals, and in the absence of histopathological changes, these variations were considered to be fortuitous.

In the animals sacrificed at the end of the treatment-free period, there were no changes in the mean organ weights which were considered to be associated with the test item. The mean absolute and relative weights of the thymus were lower in both sexes previously given 45 mg/kg bw/day, reaching astatistically significant level for the absolute value in males (p<0.05).
Any relationship with the treatment was considered to be unlikely since differences were due to isolated animals from control and high-dose group.

GROSS PATHOLOGY (PARENTAL ANIMALS)

There were no treatment-related macroscopic findings.

HISTOPATHOLOGY (PARENTAL ANIMALS)

At 45 mg/kg bw/day, changes were observed in the gastro-intestinal tract (forestomach, ileum and jejunum) and in the mesenteric lymph node of male and female rats.
In the forestomach, pronounced signs of irritation characterized by acanthosis with hyperkeratosis, ulceration and inflammation were observed in males given 45 mg/kg/day. These changes were considered to be adverse. In females, only minimal acanthosis was observed in one animal. None of these changes were observed at 5 or 15 mg/kg bw/day.
At 45 mg/kg bw/day, enlarged foamy macrophages were observed in the lamina propria of the ileum of all but one animals and in the jejunum of only one animal. None of these changes were observed at 5 or 15 mg/kg bw/day.
Enlarged foamy macrophages (occasionally forming aggregates) associated with dilation of sinuses were observed in the mesenteric lymph nodes with a dose-related trend at all dose-levels in females and from 15 mg/kg bw/day in males . In males given 45 mg/kg bw/day, this was associated with granulocytes. In the absence of degenerative changes, this change was considered to be non adverse.

After the treatment-free period, signs of recovery were observed in animals previously given 45 mg/kg bw/day, particularly in the forestomach where only minimal acanthosis was observed in a single male. Enlarged macrophages (occasionally forming aggregates) were observed in the ileum and the mesenteric lymph nodes, but with a slightly lower incidence and/or severity than at the end of the treatment period suggesting a partial recovery.

OTHER FINDINGS (PARENTAL ANIMALS) See section 7.5.1 : CIT 2010a / K1 KS / Repeated dose toxicity: oral

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on the reduction of body weight and body weight gain, the significant changes in hematological and blood biochemical parameters observed in males treated at 45 mg/kg/day.

2

Dose descriptor:

NOEL

Remarks:

reproductive performance (mating and fertility)

Effect level:

45 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No relevant effect at maximal tested dose-level

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Few pups were found dead or were cannibalized. There were not significantly more pup mortalities in the test item-treated groups than in the control group.

CLINICAL SIGNS (OFFSPRING)
Generalized pallor, thin appearance and/or coldness to the touch were observed in a few pups per group, including the controls. There was no treatment relationship.

BODY WEIGHT (OFFSPRING)
There were no effects on mean pup body weight on post-natal day 1 but the pups from the group treated at 45 mg/kg/day had lower mean body weight gains between post-natal day 1 and post natal day 5 (-14% for the males and -12% for the females). There were no effects at 5 or 15 mg/kg/day. (see table 6 in remarks on results)

GROSS PATHOLOGY (OFFSPRING)
No test item treatment-related gross external abnormalities were observed on pups from any groups.

OTHER FINDINGS (OFFSPRING)
There were no relevant differences in the percentage of male pups per group when compared with the controls.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

toxic effects on progeny

Generation:

F1

Effect level:

45 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The lower pup body weight gain at this dose-level was only slight.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of clinical signs

Dose-level (mg/kg/day)	0	5	15	45
Ptyalism		1♂	10♂, 10♀	15♂, 15♀
Loud breathing				5♂, 6♀
Round back				2♂, 3♀
Piloerection				2♂, 4♀
Cold to the touch				1♂
Hypoactivity				1♂, 6♀
Half-closed eyes				3♂, 6♀

Table 2: Summary of body weights and body weight gains (in g)

Sex	Male				Female
Dose-level (mg/kg/day)	0	5	15	45	0	5	15	45
Treatment period
Day 1	412	411	418	408	233	230	228	229
Day 15	452	462	473	425	253	253	256	246
Day 36	499	528	541*	470	268			271
Days 1-8	+22	+26	+26	+1#	+8	+13	+17**	+9
Days 8-15	+19	+25	+30*	+16	+11	+11	+11	+9
Days 1-36	+87	+117*	+123*	+61	+40			+37
Treatment-free period
Day 43	554			500*	279			286
Day 64	609			550	293			311
Days 36-64	+74			+73	+25			+40*

Table 3: Mating data

Dose-level (mg/kg/day)	0	5	15	45
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
Number of males mated	10	10	10	10
Number of females mated	10	10	10	10
Mean number of days taken to mate	1.9	3.2	2.9	3.4

Table 4: Fertility data

Dose-level (mg/kg/day)	0	5	15	45
Number of mated females	10	10	10	10
Number of pregnant females	10	10	10	9

Table 5: Delivery data

Dose-level (mg/kg/day)	0	5	15	45
Mean duration of gestation (days)	21.2	21.6	21.5	21.4
Mean number ofcorpora lutea	15.2	15.2	15.8	16.2
Mean number of implantations	14.7	14.3	15.6	15.9
Mean pre-implantation loss (%)	3.8	6.4	1.1	1.8
Mean number of pups delivered	13.5	11.6	14.5	14.7
Mean post-implantation loss (%) (calculated manually)	7.5	20.3	6.8	7.8

Table 6: Pup body weight

Sex	Male				Female
Dose-level (mg/kg/day)	0	5	15	45	0	5	15	45
Body weight (g)
Day 1	7.2	7.2	7.0	7.0	6.8	7.1	6.6	6.5
Day 5	11.5	11.9	11.3	10.8	10.9	12.1	10.9	10.1
Body weight change (g)
Days 1-5	+4.4	+4.7	+4.3	+3.8	+4.2	+5.0	+4.3	+3.7

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity with respect to systemic toxic effects was considered to be 15 mg/kg/day because of the reduction of body weight and body weight gain, the significant changes in hematological and blood biochemical parameters observed in males treated at 45 mg/kg/day. The No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 45 mg/kg/day and the NOAEL for toxic effects on progeny was 45 mg/kg/day since the lower body weight gain at this dose-level was only slight.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for the females, through gestation until day 5 post-partum. An additional satellite group was included in the control and high-dose groups for observation of reversibility, persistence or delayed occurrence of systemic/irritative effects for 4‑weeks post-treatment.

This study provides initial information on possible toxicological effects likely to arise from repeated exposure over a relatively limited period of time and on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

The study was conducted according to OECD Guideline 422 and EPA guideline OPPTS 870.3650 and was in compliance with the principles of Good Laboratory Practice regulations.

Three groups of Sprague-Dawley rats received the test substance daily, by gavage, before mating and through mating and, for the females, through gestation until day 5post-partum. The dose-levels were 5, 15 or 45 mg/kg bw/day. The low- and intermediate-dose groups were each composed of 10 males and 10 females and the high-dose group was composed of 15 males and 15 females. In the high-dose group, the first 10 animals/sex having been mated were sacrificed at the end of the treatment period ; the last 5 animals/sex not mated were dosed for 4 weeks and then retained for a 4-week treatment-free period.

Another group of 15 males and 15 females received the vehicle, sesame oil, alone, under the same experimental conditions and acted as a control group. As for the high-dose group, the first 10 animals/sex were mated and sacrificed at the end of the treatment period and the last 5 animals/sex were retained for a 4-week treatment-free period. The dosing volume was 5 mL/kg bw/day.

 In all animals from the principal and satellite groups, clinical signs and mortality were checked at least once daily during the treatment period and detailed clinical examinations were performed once before the beginning of the treatment period and then once a week until the end of the study. Body weight and food consumption were recorded weekly and at designated intervals throughout gestation and lactation. A Functional Observation Battery, including assessment of responses, reflexes, forelimb grip strength, landing foot splay and rectal temperature, was performed on the first five males and the first five females to deliver from each principal group at the end of the study. At the end of the Functional Observation Battery, motor activity was measured over a 1-hour period.

Blood and urine samples were taken from the first five males and the first five females to deliver from each principal group at the end of the treatment period for analysis of hematology, blood biochemistry and urine parameters. In addition, hematological and selected blood biochemical parameters were measured at the end of the treatment-free period in the animals of the satellite group.

The first ten animals/sex/group were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until day 5post-partum. The total litter sizes and numbers of pups of each sex were recorded after birth. Pups were observed daily for clinical signs and pup body weights were recorded on days 1 and 5 post-partum.

 In the principal groups, males were sacrificed after completion of the mating period and females on day 6 post-partum. Animals from the satellite group were sacrificed at the end of the treatment-free period. All animals were subjected to a complete macroscopic post-mortem examination with a careful examination of the stomach lining due to the strong irritant / corrosive properties of the test item. A microscopic examination was performed on selected organs of the first five males and the first five females to deliver from the control and high-dose groups sacrificed at the end of the treatment period, on the one non-pregnant female and on all macroscopic lesions. In addition, the forestomach, ileum and mesenteric lymph nodes from the low- and intermediate-dose groups sacrificed on completion of the treatment period and from the control and high-dose groups sacrificed at the end of the treatment-free period were examined.

  Pups were sacrificed on post-natal day 5 and were carefully examined for gross external abnormalities and macroscopic abnormalities. Found dead and prematurely sacrificed pups were also examined for gross external and macroscopic abnormalities.

There were no unscheduled mortalities. Hypersalivation was observed in all males and females treated at 15 or 45 mg/kg bw/day. In addition, some males and females treated at 45 mg/kg bw/day had loud breathing, round back, piloerection, hypoactivity and half-closed eyes on occasions during the study. No relevant clinical signs were observed at 5 mg/kg bw/day.

Males treated at 45 mg/kg bw/day had statistically significantly lower mean body weight gains and food consumption in the first week of treatment (seven males lost weight). The deficit was not recouped during the treatment period or during the treatment-free period. Females treated at 45 mg/kg bw/day also had statistically significantly lower mean food consumption in the first week of treatment.There were no effects at 5 or 15 mg/kg bw/day.

There were no effects on mating, fertility or delivery at any dose-level. There were no increases in pup mortality in the test item-treated groups and there were no relevant clinical signs or gross abnormalities in the pups. Mean pup body weight gain between post-natal days 1 and 5 at 45 mg/kg bw/ day was lower for both sexes when compared with controls. There were no effects at 15 or 15 mg/kg bw/ day.

Laboratory investigations showed that males treated at 45 mg/kg bw/day had a significant leucocytosis and a slight anemia together with a decrease in protein and albumin concentrations but at the end of the treatment-free period, all parameters were comparable with controls. There were no treatment-related effects on hematological and biochemical parameters at 5 or 15 mg/kg/day in males or in females at any dose-level.

No treatment-related effects on organ weights and no particular macroscopic findings were observed.

Microscopic examination revealed enlarged foamy macrophages graded as minimal or slight in the ileum of males and females treated at 45 mg/kg bw/day. No particular findings were found in the ileum of animals treated at 5 or 15 mg/kg bw/day. Enlarged foamy macrophages, occasionally forming aggregates at high dose and associated with dilation of sinuses, were also observed in the mesenteric lymph nodes with a dose-related trend at all dose-levels in females and from 15 mg/kg bw/day in males. At the lower doses, the finding was marginal: 1 male treated at 15 mg/kg bw/day and 1 female treated at 5 mg/kg bw/day had a minimal infiltration in the mesenteric lymph nodes. As no degenerative changes were associated, this effect was therefore considered to be non-adverse. Partial recovery was observed by the end of the treatment-free period.

Symptoms related to the strong irritant / corrosive nature of the test substance were observed in the forestomach of males treated at 45 mg/kg bw/day at the end of the treatment period. Males rats showed acanthosis and edema of the forestomach mucosa and ulcerations and hyperplasia in the forestomach.This was considered to be treatment-related and adverse. At the end of the treatment-free period, only 1/5 males had minimal acanthosis indicating recovery.

In conclusion, clinical signs of poor condition were observed in some males and females at 45 mg/kg bw/day. Week 1 body weight gain was markedly lower in males (some males lost weight) and food consumption was reduced in males and females. Males did no

recoup the low body weight gain and mean body weight remained lower at the end of the treatment-free period. There were no effects on mating, fertility or delivery and pups showed no effects other than a slightly lower mean body weight gain from post-natal day 1 to 5. A significant leucocytosis and a slight anemia together with a decrease in protein and albumin concentrations were observed in males but all showed recovery by the end of the treatment-free period. There were no treatment-related macroscopic findings or treatment-related effects on organ weights. At microscopic examination, the strong irritating/corrosive properties of the test item were confirmed through the findings in the forestomach of all males. These findings were regarded as an irritant/corrosive effect and not as symptoms of a cumulative-systemic toxicity but were considered to be adverse. Both males and females had enlarged foamy macrophages in the ileum and mesenteric lymph nodes. They probably represent histiocytes containing test-item lipid complexes which are poorly degraded by lysosomal enzymes. As no degenerative changes were associated, this effect was therefore considered to be non adverse. Recovery was observed at the end of the treatment-free period in all organs, particularly in the forestomach, where only minimal acanthosis was observed in one male.

 At 5 and 15 mg/kg/day, there were no adverse clinical signs and no effects on body weight or food consumption. There were no effects on mating or fertility and there were no effects on the pups after birth. None of the hematological or blood biochemical parameters were affected by treatment and no abnormal responses, reflexes or behavior were observed during the Functional Observation Battery. There were no treatment-related macroscopic findings and no treatment-related effects on organ weights.There were no particular findings in the forestomach and ileum at microscopic examination. Mesenteric lymph nodes revealed only minimal or slight enlarged foamy macrophages in three females and one male treated at 15 mg/kg/day and in one female treated at 5 mg/kg/day.

 Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity with respect to systemic toxic effects was considered to be 15 mg/kg/day because of the reduction of body weight and body weight gain, the significant changes in hematological and blood biochemical parameters observed in males treated at 45 mg/kg/day.

The No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 45 mg/kg/day and the

No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was 45 mg/kg bw/ day since the lower body weight gain at this dose-level was only slight.