1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C18(unsaturated) acyl derivs., hydroxides, inner salts

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

The test material corresponded to the approximately 45% of the registration substance in water.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanaminium,3-amino-N-(carboxymethyl)-N,N-dimethyl-,N-C18(unsaturated) acyl derivs., hydroxides, inner salts

IUPAC Name:

1-Propanaminium,3-amino-N-(carboxymethyl)-N,N-dimethyl-,N-C18(unsaturated) acyl derivs., hydroxides, inner salts

Test material form:

liquid

Specific details on test material used for the study:

The test material corresponded to the approximately 45% of the registration substance. The given dose refers to the amount of the registration substance.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities. The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 15-16 wks;
- Weight at study initiation: Males: 346.45 to 426.47 g; Females: 210.37 to 262.30 g;

- Housing:
Pre-mating: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes.

Mating and post-mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories (Envigo), P.O. Box 44220, Madison, WI 53744-4220, was provided ad libitum to animals.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limited., Mumbai 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: Start: 20 November 2016 End: 24 November 2016

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20−25°C
- Humidity (%): 53−68%
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle
IN-LIFE DATES: From: 25 November 2016 To: 02 March 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% Carboxymethylcellulose sodium salt (medium viscosity) in Milli-Q® Water was used to prepare the dose formulations as the same vehicle was used in the 14 day repeated dose toxicity study (N2857) and in dose range finding study for reproduction / developmental toxicity screening test by oral gavage in Wistar rats (Study No.N2856) with the same test item.

Details on mating procedure:

- M/F ratio per cage: 1 : 1
- Length of cohabitation: 21 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [ No]
- After successful mating each pregnant female was caged (how): 1 per cage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during week 4 of the treatment and analysed in-house. Samples were drawn from top, middle and bottom layers of each preparation for treatment groups in duplicate sets and from middle layers of preparation for control group (three samples corresponding to each treatment group).
The analysis was done as per the method validated under Advinus Study No.: G11354. One set of samples were analysed for concentration (a.i) analysis.
The unused back up samples was disposed as analysis results of the first set of samples were within the acceptable limits.

Duration of treatment / exposure:

Males: The dose formulation was administered orally by gavage to specific group of rats at approximately the same time each day (varying by ± 3 hours), for a period of 39 days (which includes two weeks prior to mating, during the mating period and post mating) after which they were sacrificed after overnight fasting.
Females: The dose formulation was administered orally by gavage to the specific group of rats at approximately the same time each day (varying by ± 3 hours), two weeks prior to the mating period and was continued through mating, pregnancy and up to LD 13. On LD 14, the females were sacrificed after overnight fasting.
The animals in the vehicle control group were handled in an identical manner to the treatment group and were administered vehicle only.
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control group at an equivolume of 10 mL/kg Bwt.

Frequency of treatment:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study: [15 to 16] weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the DRF (Study No. N2856)
- Rationale for animal assignment (if not random):
Grouping was done by the method of body weight stratification and distribution. On the day of randomization, based on the given temporary animal identification number, each animal with normal oestrous cyclicity (4-5 day cycle) was weighed and the corresponding body weights was recorded.
The body weight recorded was stratified in ascending order.
Statistical analysis and ensured that the weight variation was minimal and inter group variation did not exceed ± 20% of the mean body weight in each sex. Rats with extreme body weights were discarded. Grouping was done one day prior to initiation of the treatment.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily once
- Cage side observations checked.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular 4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [Yes]
- If yes, maximum of [8] pups/litter ([4]/sex/litter as nearly as possible); excess pups were killed and discarded.

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, anogenital distance (AGD), presence of nipples/areolae in male pups, other:] Yes

GROSS EXAMINATION OF DEAD PUPS:
[Yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the Guideline were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated the Guideline were prepared for microscopic examination and weighed, respectively.

Statistics:

Statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Version 12.0. All data of quantitative variables like body weight, food consumption, oestrous cycle length, hormone levels, ano-genital distance, ano-genital index and organ weights and organ weight ratios data were tested for homogeneity of variances (Levene’s test) within the group before performing One-Way Analysis of Variance (ANOVA). When the data found to be non-optimal (non-normal or heteroschedastic), ANOVA was done using suitable transformation. Comparison of means between treatment groups and vehicle control group was done using Dunnett’s test when the overall treatment ‘F’ test is found significant.
Post implantation loss (%), no. of implantations, pre-coital interval (days), mean litter size, sex ratio and gestation length (days) were analysed after suitable transformation (√ x + ½) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was performed for testing the differences found significant.
Z test was performed for testing the differences in proportions for mating, fertility and survival indices.
Data captured using Provantis™ for the parameters laboratory Investigations - Haematology (Coagulation tests PT and APTT which was entered retrospectively in ProvantisTM) and Clinical Chemistry was analyzed using built-in statistical tests
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
+/-: Significantly higher (+)/lower (-) than the vehicle control group

Reproductive indices:

Reproductive Performance Data of Parents
a. Male mating index (%) = (Number of males with evidence of mating / Number of males cohabited) x 100

b. Male fertility index (%) = (Number of males siring a litter / Number of males cohabited) x 100

c. Female mating index (%) = (Number of females mated / Number of females cohabited) x 100

d. Female fertility index (%) = (Number of pregnant females (confirmed at necropsy) / Number of females used for mating) x 100

e. Mean number of implantations/group = (Total number of implantations / Total number of pregnant animals)

f. Post implantation loss (%) = (Number of implantations - Number of live pups / Number of implantations) x 100

Offspring viability indices:

Litter Data
a. Mean litter size per group = (Total Number of pups / Total Number of littered animals)

b. Mean viable litter size = (No. of viable pups on Day 1 / No. of females littered)

c. Live birth index (%) = (No. of viable pups born (at first observation) / Total no. of pups born (at first observation)) X 100

d. Day 4 survival index (%) = (Number of viable pups on lactation Day 4 / Number of viable pups born) x 100

e. Sex Ratio (%) = (No. of male pups born / Total No. of pups born) x 100

f. Ano-genital Distance Ratio (mm/g1/3) = (Ano-genital distance / Cube root of body weight)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed during the treatment period at all the treated groups in both sexes.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two females at dose of 800 mg/kg bw/day died due to dystocia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights and body weight gains were unaffected throughout the treatment period at 100 and 300 mg/kg Bwt/day doses when compared to vehicle control in males. In females the mean body weights and net body weight gains were comparable to vehicle control group during two weeks pre-mating period.
At 800 mg/kg Bwt/day, the mean body weights on Weeks 1 and 2 (-7.8 to -11.8%, respectively) and the body weight gain were significantly lower in males. In females the mean body weights and net body weight gains were comparable to vehicle control group during two weeks pre-mating period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption was not altered by the treatment in throughout the treatment period in males and two weeks pre-mating period in females at 100 and 300 mg/kg Bwt/day doses, when compared to vehicle control. However, an incidence of significantly lower food consumption during week 1 at 300 mg/kg Bwt/day dose was observed. This decrease in food consumption was considered to be toxicologically not significant as the mean body weights were comparable to control.
At 800 mg/kg Bwt/day, the food consumption was significantly lower on Week 1 in males, with a reduction of 52.2%, when compare to control. In females the food consumption was comparable to vehicle control group during two weeks pre-mating period in females. However, an incidence of significantly lower food consumption during week 1 at 800 mg/kg Bwt/day dose in females was observed. This decrease in food consumption was considered to be toxicologically not significant as the mean body weights were comparable to control.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in the haematology and coagulation parameters analysed across the groups in female rats.
All the statistical significant changes observed in haematology parameters were considered as incidental in nature, due to the minimal magnitude without dose progression and also the values were within the range of biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes observed in the clinical chemistry parameters analysed across the groups in female rats.
All the statistical significant changes observed in clinical chemistry parameters across the treatment groups were considered as incidental in nature, due to the minimal magnitude without dose progression and also the values were within the range of biological variation.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related histopathological changes in the reproductive organs in both males and females.
The staging of spermatogenesis did not reveal any stage specific changes. The spermatogenic cycle observed in the different seminiferous tubules of testes were complete and none of the stages of spermatogenesis were arrested in all the animals examined.
There was no test item-related microscopic changes observed in thyroid gland of parents and pups at all the dose levels tested.
Microscopically, ulceration, hyperkeratosis and epithelial hyperplasia were observed in non-glandular mucosa of stomach at ≥ 300 mg/kg/day in both sexes and considered test item-related change.
All the other microscopic findings observed across the groups in male and female rats were considered as incidental/spontaneous changes and not related to administration as the findings were distributed randomly across the groups and normally observed at these age group animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Two females at 800 mg/kg bw/day died due to dystocia. One female at 300 mg/kg bw/day died due to dystocia, but the incidence (one out of ten) was within historical range.

Details on results (P0)

The purpose of this study in Wistar Rats was to generate limited information concerning the effects of C18unsat-AAPB on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.
The test item was weighed and dissolved in vehicle i.e., 0.5% Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q Water and administered by oral gavage at the dose levels of 100 (G2), 300 (G3) and 800 (G4) mg/kg Bwt/day to male and female Wistar rats, at the dose volume of 10 mL/kg Bwt/day. Similarly, vehicle was administered to rats in the vehicle control group. Each group consisted of 10 male and 10 female rats. The prepared dose formulations were administered once daily to specific group of rats prior to mating, during mating and post-mating periods for males, prior to mating, during conception and pregnancy and after delivery up to Lactation Day (LD) 13 for females
The identity of C18unsat-AAPB was provided by the study Sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of the test item in the vehicle was established at 1 and 200 mg/mL under Advinus Study No. G11354. Based on the results, the test item was stable and homogenous in the vehicle (0.5 % Carboxymethylcellulose sodium salt (medium viscosity) in Milli-Q® water) up to 24 hours when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during 2nd month of the treatment period. The results indicated that the analysed concentrations were within ± 20% of variations from the nominal concentrations.
All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period. Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4, 7 and 13. The number, weight, survival and mortality of pups were recorded during the lactation period. The ano-genital distance of each pup was measured on LD 4. All the survived male pups were examined for the appearance of nipples/areolae on post-natal day (PND 13). Prior to necropsy from males, on LD 4 and 13 from available pups, the blood samples were collected for thyroid hormone analysis. The animals were subjected to detailed necropsy at sacrifice and study plan specified tissues were collected. From female rats (dams), the blood was collected on LD 13 prior to necropsy. In addition, the clinical laboratory investigations such as haematology, coagulation and clinical chemistry analysis were performed prior to necropsy from surviving females. A gross necropsy was performed on all dams on LD 14 and study plan specified tissues were collected. All the surviving pups were sacrificed on LD 13 and thyroid gland from available one male and one female pup from each litter was collected for histopathological examination.
Tissues collected from all animals in the control and high dose groups were examined microscopically for histopathological changes. Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. All gross lesions were examined in all the groups. The reproductive organs of animals failing to mate were examined in all the dose groups.
Under the experimental conditions employed, the following results were obtained:
• There were no treatment-related clinical signs observed at any of the doses tested. Two females died due to dystocia at the 800 mg/kg Bwt/day.
• The body weights and food consumption were unaffected by the treatment in males at 100 and 300 mg/kg Bwt/day and all the tested doses in females. At 800 mg/kg Bwt/day, the decreased body weights during initial 2 weeks of treatment period were associated with lower food consumption.
• The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the doses tested.
• Treatment had no effect on pre-coital time or gestation length, oestrous cycle length at all the tested doses. No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested. The survival indices were not altered by the treatment at 100 and 300 mg/kg Bwt/day doses.
• At 800 mg/kg Bwt/day, the treatment significantly decreased the mean weight of pups on LD 4. The 24 hour and Day 4 survival indices were significantly lower when compared to vehicle control due to the higher number of pups dead/cannibalised from LD 1 to 4.
• There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups.
• No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the vehicle control group.
• The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.
• There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in males at 100 and 300 mg/kg Bwt/day and all the tested doses in females. At 800 mg/kg Bwt/day, the treatment decreased the terminal body weights in males. Gross examination of pups on LD 13 did not reveal any gross changes.
• The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and in male pups were not significantly different from the control. In female pups, increase in thyroid stimulating hormone (TSH), decrease in thyroxin hormone (T4) level and increased thyroid gland weight (on LD 13 after birth) was considered as a test item-related non adverse change in the absence of correlating histopathological changes in thyroid gland at 300 mg/kg Bwt/ day.
• There were no test item-related changes in haematology, coagulation, clinical chemistry in females.
Gross and histopathology: Macroscopically, multiple foci / thickening in non-glandular mucosa of stomach were microscopically associated with ulceration, hyperkeratosis and epithelial hyperplasia in both sexes at 300 mg/kg Bwt/day. At 800 mg/kg Bwt/day grossly multiple foci / thickening in non-glandular mucosa of stomach were microscopically associated with ulceration, hyperkeratosis and epithelial hyperplasia in both sexes at 800 mg/kg/day.

There were no test item-related gross and microscopic changes observed in the reproductive organs and thyroid gland of male and female parents at all the tested doses.

No Observed Adverse Effect Level
Daily oral (gavage) administration of C18unsat-AAPB to male and female Wistar rats at dose levels of 100 and 300 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery did not induce any adverse effects on fertility, reproductive performance and on the offspring parameters.
Considering the dystocia deaths, lower pup weight on LD 4, lower 24 hour and Day 4 survival index at 800 mg/kg Bwt/day dose group, the no observed adverse effect level (NOAEL) for maternal toxicity, reproductive toxicity and developmental toxicity of C18unsat-AAPB is considered to be 300 mg/kg Bwt/day.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

gross pathology

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

reduced survival index at 800 mg/kg bw/day after birth

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced pup body weight on LD4

Ophthalmological findings:

no effects observed

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Table 1: Summary of Body Weights of Males

Doses [mg/kg /day]		Body weight of males [g]; n = 10							Body weight gain; week 1-6
		1d	1w	2w	3w	4w	5w	6w
0	Mean	391.27	396.65	406.83	409.94	417.46	425.23	429.53	38.26
	S.D.	24.00	29.22	30.85	32.20	34.69	36.25	38.84	16.71
100	Mean	392.67	400.48	413.99	417.85	423.09	429.75	432.84	40.16
	S.D.	23.14	26.12	27.09	29.86	29.76	31.55	31.17	12.18
300	Mean	392.33	399.06	407.39	411.80	421.86	422.36	423.90	31.57
	S.D.	21.90	24.70	26.78	29.92	31.45	33.21	35.38	19.30
800	Mean	393.46	349.59	374.92	380.00	393.11	397.13	396.20	2.74
	S.D.	20.33	25.61	22.55	19.44	23.38	23.93	23.08	24.09

Table 2: Summary of Body Weights of Females; Pre-mating

Doses [mg/kg /day]		Body weight [g]; n = 10			Body weight gain;  n = 10
		1d	1w	2w
0	Mean	239.06	238.71	238.60	-0.47
	S.D.	15.13	14.32	13.90	3.27
100	Mean	238.08	236.96	240.71	2.63
	S.D.	14.87	13.27	15.16	4.73
300	Mean	238.91	235.72	240.91	2.00
	S.D.	11.42	14.36	14.46	7.14
800	Mean	239.16	233.93	244.66	5.51
	S.D.	12.27	11.47	14.13	8.91

Table 3: Summary of Body Weights of Females; Gestation

Doses [mg/kg /day]		Body weight [g]				Body weight gain
		GD1	GD7	GD14	GD20
0	Mean	245.54	264.95	288.18	334.81	89.27
	S.D.	11.22	15.05	16.54	21.99	19.50
	n	9	9	9	9	9
100	Mean	241.96	259.57	283.85	334.01	92.05
	S.D.	18.00	19.19	19.76	24.13	17.40
	n	8	8	8	8	8
300	Mean	240.04	260.52	283.40	327.90	87.86
	S.D.	15.31	15.33	18.53	31.60	22.37
	n	9	9	9	9	9
800	Mean	240.02	255.16	277.36	321.17	81.15
	S.D.	12.51	13.07	14.88	23.30	16.93
	n	8	8	8	8	8

Table 4: Summary of Body Weights of Females; Lactation

Doses [mg/kg /day]		Body weight on Day [g]			Weight change during
		LD0	LD4	LD13	LD 0-4	LD 4-13	LD 0-13
0	Mean	258.05	266.99	274.78	8.94	7.80	16.73
	S.D.	16.16	14.85	14.16	8.67	7.39	10.18
	n	9	9	9	9	9	9
100	Mean	257.40	260.95	273.09	3.56	12.14	15.70
	S.D.	14.72	20.27	17.61	13.33	7.78	9.10
	n	9	9	9	9	9	9
300	Mean	254.81	268.91	274.71	14.11	7.76	22.94
	S.D.	21.61	19.03	21.14	9.05	4.97	9.56
	n	8	8	6	8	8	6
800	Mean	244.26	248.57	282.48	0.52	28.96	26.68
	S.D.	15.44	16.76	3.27	13.52	6.35	14.36
	n	6	5	2	5	2	2

Table 5. Summary of reproductive parameters

Dose (mg/kg bw)	0	100 mg/kg/day	300 mg/kg/day	800 mg/kg/day
Number of females paired	10	10	10	10
Number of females mated (confirmed by vaginal smear)	10	9	9	10
Number of females pregnant (confirmed at littering/necropsy)	9	9	9	8
Dystocia death	0	0	1	2
Number of females with live litters	9	9	8	6
Precoital interval (days)	3.1 ± 1.0(n= 10)	2.8 ± 1.0(n= 9)	1.7 ± 0.9(n= 9)	3.2 ± 1.7(n = 10)
Gestation length (days)	22.9± 0.6(n= 9)	22.8± 0.7(n=8^a)	22.6 ± 0.7(n= 8)	22.7 ± 0.5(n = 6)
Implantation sites	10.1 ± 2.7 (n= 9)	10.1 ± 3.1 (n=9)	9.7 ± 3.3 (n= 9^b)	10.5 ± 2.1 (n=8^b)
Mean viable litter size	8.6 ± 2.1(n= 9)	8.4 ± 2.5(n=9)	8.5 ± 3.4(n= 8)	9.8 ± 2.5(n= 6)
Number of pups postnatal^c
Total number born	80	77	68	60
Total number born alive	77	76	68	59
Total number alive on LD 4	69	74	57	26
Total number after culling on LD4	61	64	48	20
Total number alive on LD13	53	53	40	10
Total number death LD 0-4	11	3	11	34
Total number death LD 4-13	0	0	3	10

a. One value missing

b. Includes the values obtained from the animals with dystocia death

c. Excluding the values obtained from the animals with dystocia death

Table 6: Summary of mean body weight (g) of pups during the lactation period

Doses [mg/kg /day]		Mean body weight of male pups on Day			Mean body weight of male pups on Day			Mean body weight of male pups on Day
		0	4	13	0	4	13	0	4	13
0	Mean	6.29	10.45	27.75	6.08	10.16	27.37	6.18	10.26	27.47
	S.D.	0.51	1.33	3.87	0.58	1.23	3.36	0.53	1.24	3.42
	N	9	9	9	9	9	9	9	9	9
100	Mean	6.29	9.92	26.36	5.92	9.57	25.08	5.92	9.57	25.08
	S.D.	0.85	2.28	7.63	0.73	2.01	5.09	0.73	2.01	5.09
	N	9	9	9	9	9	9	9	9	9
300	Mean	5.94	8.79	24.97	5.82	9.11	24.11	5.84	8.69	24.53
	S.D.	0.63	1.88	4.46	0.73	1.80	5.03	0.65	1.99	4.67
	N	8	7	6	7	6	6	8	7	6
800	Mean	6.00	5.72	19.85	5.71	6.56	24.05	5.80	6.68	24.30
	S.D.	0.21	0.98		0.35	2.41	7.35	0.28	2.35	6.99
	N	6	3	1	6	4	2	6	4	2

Applicant's summary and conclusion

Conclusions:

The registration substance was investigated for its reproduction toxicity according the Guideline OECD 421. The NOAEL of 300 mg/kg bw/day was obtained for systemic toxicity, reproduction performance and developmental toxicity.

Executive summary:

The registration substance was investigated for its reproduction toxicity according the Guideline OECD 421. Rats were treated up to dose 800 mg/kg bw, once per day, for two weeks prior to mating,throughout mating, gestation, and lactation up to postnatal day 13. At 800 mg/kg bw/day, reduced body weight for males and multiple foci/thickening in non-glandular mucosa in stomach in males and females were found. Further, dystocia and increased postnatal mortality were observed at this dose, which is likely secondary to systemic toxicity.

The NOAEL of 300 mg/kg bw/day was established for systemic toxicity, reproduction performance and developmental toxicity.