2,2'-(cyclohexylimino)bisethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The submission substance was tested in a guideline conform reproductive toxicity screening study according to OECD TG 421. No relevant effects on fertility / reproduction or development were observed. The parental NOAEL in this study was 75 mg/kg body weight (based on reduced body weights, inflammatory changes and mortality at higher dose levels evidenced by repeated dose toxicity studies) and the NOAEL for fertility / reproduction was also 75 mg/kg body weight. The submission substance is therefore considered to not exert reproductive toxic (fertility) effects.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP

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:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar rat, Crl: WI(Han) (SPF), Charles River, Germany
- Age at study initiation: 9-10 weeks old (males and females)
- Weight at study initiation: 249-288 g (males); 185-213 g (females)
- Fasting period before study: over night
- Housing: individual housing in IVC cages, type III H
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hourd light / dark

Route of administration:

oral: gavage

Vehicle:

water

Details on mating procedure:

Animals were paired in the ratio 1:1 until evidence of copulation was observed. The females were removed and housed individually when:
- vaginal smear was sperm positive
- a copulation plug was observed
The day of vaginal plug and/or sperm was considered as day 0 of gestation.
All dams were allowed to give birth and rear their litter (F1 pubs) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pubs.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males were dosed 28 days
Females were dosed 54 days

Frequency of treatment:

once per day, 7 days per week

Remarks:

Doses / Concentrations:
0 mg/kg
Basis:
actual ingested

Remarks:

Doses / Concentrations:
12 mg/kg
Basis:
actual ingested

Remarks:

Doses / Concentrations:
30 mg/kg
Basis:
actual ingested

Remarks:

Doses / Concentrations:
75 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on dose-range finder
- Rationale for animal assignment (if not random): random

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: before assignment to groups; males weekly thereafter; females weekly during pre-mating period and on gestation day 0, 7, 14, 20 as well as on post-natal day 4

Sperm parameters (parental animals):

Detailed qualitative examination of the testes including the tubular stages of the spermatogenic cycle

Litter observations:

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

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

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
- cervix, coagulating gland, epididymis, ovary, prostate, seminal vesicle, testis, uterus, vagina

Postmortem examinations (offspring):

SACRIFICE
- on day 4 post natal

GROSS NECROPSY
- performed on all animals

Reproductive indices:

copulation index, delivery index, fertility index, viability index

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall effects

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 75 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall effects

Reproductive effects observed:

not specified

Conclusions:

Based on the results of this study, the NOAEL for reproductive toxicity of the test item is equal or greater 75 mg/kg body weight per day.

Executive summary:

Four groups comprised of 10 adult males and 10 non pregnant nulliparous female rats (Wistar Crl:WI) were dosed daily by oral gavage with 12, 30 and 75 mg/kg body weight per day of the test item at dose volume of 5 mL/kg body weight using sterile water as vehicle. Doses were selected based on a dose-range finder study. Control animals were treated identically with the vehicle alone. After 14 days of treatment to both male and female, animals were paired (1:1) till the evidence of mating in the form of sperm positive vaginal smears. Males and females were sacrificed on treatment day 29 and post natal day 4 respectively and subjected to necropsy.

No test item related clinical signs and mortalities were observed in both males and females. Body weight development and food consumption was not affected. Group mean litter weight, total litter weight as well as male and female litter weight on post natal day 0 and 4 was unaffected. No treatment related effect was observed on precoital interval and duration of gestation. Pre and post natal data like group mean number of corpora lutea, percent preimplantation loss and post implantation loss remained unaffected due to treatment when compared with controls. No treatment related effects were observed on reproductive indices like coagulation index, delivery index, fertility index and viability index and there were no effects on litter data like number of males and females, sex ratio and still birth. Survival of pubs from post natal day 0 to 4 remained unaffected and treatment related gross external findings were not observed.

With regard to organ weights, in males a statistical significant decrease in absolute right testes weight in LD group and a decrease in absolute epididymides (right, left and total weight) and relative total epididymides weight in LD and HD group when compared with the controls were revealed. However, since no dose relationship was established and no histopathological changes were observed, no toxicological significance is attributed to these findings. At necropsy of all male and female animals, no macroscopic changes were observed and no treatment-related histopathological findings of reproductive organs were revealed.

A detailed qualitative examination of the testes taking into account the tubular stages of the spermatogenic cycle, revealed no abnormal pathological findings.

In conclusion, the repeated administration of the test item to male animals for 28 days and female animals for a maximum of 54 days revealed no significant toxicological findings and mortalities. Reproductive toxic effects were not revealed. Based on the data, the no observed adverse effect level (NOAEL) for maternal toxicity was equal or greater 75 mg/kg body weight per day. The no observed effect level (NOEL) for reproductive toxicity was equal or greater 75 mg/kg body weight per day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline study according to GLP available. No derivations and/or confounders. Klimisch rating 1 representing reliability without restrictions. Information valid and meets data requirements.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The submission substance was tested for potential reproductive toxicity in a screening assay according to OECD TG 421 following oral administration to rats. Based on toxicity data revealed from a 14 -day repeated oral dose-range-finder toxicity study, 75 mg/kg body weight per day was selected as highest dose level based on reduced body weight gains, generally poor health status, gastrointestinal tract findings and inflammatory effects evidenced by white blood cell counts at a dose level of 125 mg/kg body weight as well as mortalities observed at 500 mg/kg body weight per day. However, the highest dose of 75 mg/kg body weight per day chosen for the reproductive screening study did not affect any relevant biological parameter, neither with regard to parental toxicity nor with respect to reproductive (fertility) toxic effects. Both, the parental NOAEL and the reproductive (fertility) NOAEL in this study was established at 75 mg/kg body weight per day. Likewise, evidence from extensive histopathological evaulation of reproductive organs including pathological sperm staging after repeated administration of the submission substance to rats for exposure periods up to 28 -days have not revealed indications of reproductive / fertility toxic effects. It can therefore be concluded that the submission substance is devoid of reproductive toxic (fertility) potential below doses causing significant maternal toxic effects.

Justification for selection of Effect on fertility via oral route:
Guideline study according to GLP with a Klimisch rating 1.

Effects on developmental toxicity

Description of key information

OECD 421:

No indications regarding developlental toxicity from an oral reproductive toxicity screening study exist. Likewise, no manifestations of adverse structural or functional changes in reproductive organs were revealed during detailed qualitative examination of the testes including the tubular stages of the spermatogenic cycle.

OECD 414:

The developmental toxicity of the registration substance was evaluated in rats in a prenatal developental toxicity study according to OECD 414. The NOAEL for maternal toxicity is 180 mg/kg/day, as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 180 mg/kg/day. The NOAEL for developmental toxicity and teratogenicity is 180 mg/kg/day, as fetal resorptions or post implantation loss were comparable to the control, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 180 mg/kg/day..

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 October 2020 to 17 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Manufactured By
Clariant Iberica Produccion, S.A.
Autovia Tarragona-Salou
3110 La Canonja, Spain

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Source: Hylasco Biotechnology (India) Pvt. Ltd.
4B, MN Park, Shameerpet Mandal,
Turkapally Village,
Medchal District-500078, Telangana, India

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulation samples were analyzed for active ingredient concentrations. The results indicated that the analyzed concentrations were within ± 10.0 % of the nominal concentration, and the relative standard deviation (% RSD) was less than 10.0 %.
Dose Level (mg/kg/day) Nominal Con.(mg/mL) Average Calculated Con.(mg/mL) Average %Recoverya, b % Relative Standard Deviationa
0 0.0 0.0 NA NA
45 4.5 4.62 - 4.50 102.7 - 100.0 0.606 - 0.958
90 9 9.24 - 8.9 102.6 - 98.87 0.595 - 0.398
180 18 18.1 - 18.0 100.5 - 99.81 0.416 - 0.287
a Results are the range of values determined from two occasions.
b Average % recovery was calculated from the nominal concentration.
NA – Not Applicable

Details on mating procedure:

The female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug we re examined in the morning hours of the subsequent day to confirm mating.

Duration of treatment / exposure:

Gestation day 5 to gestation day 19

Frequency of treatment:

Daily from gestation day 5 to gestation day 19

Duration of test:

Upto gestation day 20

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

45 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

90 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

180 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

24 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A preliminary dose range finding study (DRF) in pregnant rats was carried out using 7 rats per group with Genamin CH 020 dosed at 45, 67.5, 100 and
150 mg/kg/day along with the concurrent vehicle control group (Study number N4888). The rats were treated with the dose formulations by oral gavage at a dose volume of 10 mL/kg body weight from GD 5 to 19 and observed for clinical signs and mortality.

In the DRF study, dose levels up to 150 mg/kg/day were tolerated. No test item related clinical signs were noted at any dose. The body weight gains, and food consumption were unaffected up to the highest dose of 150 mg/kg/day. There was no fetal developmental toxicity up to the highest tested dose of 150 mg/kg/day.

Based on the results of the dose range finding study and considering results from additional repeated dose toxicity studies, the following dose levels have been selected for this definitive study in consultation with the Sponsor:

G1 - Vehicle control - 0 mg/kg/day
G2 - Low dose - 45 mg/kg/day
G3 - Mid dose - 90 mg/kg/day
G4 - High dose - 180 mg/kg/day

- Rationale for animal assignment (if not random):

During the mating period, the female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug were examined in the morning hours of the subsequent day. If sperm was detected in a vaginal smear and or if a vaginal plug was observed in the morning, the female was considered to be mated. This day was considered as Day 0 of gestation.

The females were cohabited in batches of required numbers. This procedure was continued until there were sufficient numbers of Day 0 pregnant rats for the study.

Day 0 pregnant rats obtained on each day were randomly distributed to different groups by body weight stratification method using ProvantisTM software.

After grouping and ascertaining the group mean body weight, the rats were given accession number as applicable to the group on each day of assignment.

Note: After confirming the day 0 of gestation, females were housed individually, and the unselected females and males were disposed after mating procedure.

The selected female rats were assigned to vehicle control, and three test item treatment groups.

Maternal examinations:

CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose) during treatment period
- Cage side observation checked in table 2 were included

Ovaries and uterine content:

The ovaries and uterine contents were examined after termination GD 20.
• Pregnancy status
• Gravid uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta

Blood sampling:

At caesarean section, from each rat, blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture for the determination of total Triiodothyronine (T3), Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) hormones.
Blood samples (about 1 mL from each rat) were collected in plain labelled tubes and kept on bench top for approximately 90 minutes before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4°C. The serum samples were placed in labeled plastic tubes and stored at approximately -70 °C until they were analysed.

Fetal examinations:

• Total number of fetuses
• Total number of live fetuses
• Total number of dead fetuses
• Individual fetal body weight
• Fetus sex (during visceral examination)
• External examination of fetus
• Soft tissue evaluation
• Skeletal examination
• Head examination (half the number of fetuses per litter)

Statistics:

Data captured using the ProvantisTM laboratory information management system (LIMS), parameters such as maternal body weight, body weight change, food consumption, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, Pre/post implantation loss , no. of implantations, sex ratio, Number of corpora lutea, early and late resorptions, hormone analyses (T4, T3, TSH) and the weight of thyroid gland data were evaluated using the Levene test for homogeneity of variances and the Shapiro-Wilks test for normality of distributions. Data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA). Data found to be nonhomogeneous or of nonnormal data was subjected for transformation and ANOVA was done on transformed data. When ANOVA was significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.

The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s exact test for group association.

The incidence of fetus and litter (incidence and percent) observations for skeletal observations were tested using Cochran Armitage trend test and pair wise comparison were tested by Fisher’s exact test for group association.

All hypothesis testing was carried out at the 5% (2-sided) significance level. Significant differences are designated throughout the report as below:

*: Statistically significant difference from the control group at p < 0.05

Historical control data:

Refer Annexure 8

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

There was no morbidity or mortality and the treated rats were normal throughout the experimental period at all the doses tested.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The maternal absolute mean body weights, body weight gains and adjusted/corrected body weight gain were unaffected by treatment up to the highest dose of 180 mg/kg/day. The adjusted body weight gain was significantly lower (- 12%) at 90 mg/kg/day which is considered incidental as a similar trend was not observed at the highest dose of 180 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The maternal mean food consumption was unaffected by treatment up to the highest dose of 180 mg/kg/day. At 90mg/kg/day, the food consumption during GD 17-20 was significantly lower (- 9%) which is considered incidental as a similar trend was not observed at the highest dose of 180 mg/kg/day.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in total Triiodothyronine (T3), Thyroxine (T4) or Thyroid Stimulating Hormone (TSH) hormones at any dose level.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Thyroid gland weight was unaffected by treatment. No treatment-related histopathological effects were observed in the thyroid gland.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item related findings in any of the organs on gross examination.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item related changes in organ weights and histopathology of thyroid glands from all treated groups.
The incidences of ectopic thymus and ultimobranchial cysts were randomly distributed across the groups and hence considered as incidental background findings and not related to test item administration.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone levels (T3, T4 and TSH), thyroid weights, and thyroid histology were unaffected by treatment.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions in the study

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The post implantation loss rate at 45 mg/kg/day was significantly lower which could account for the observed higher total number of fetuses in this dose group. Gross evaluation of placenta revealed no remarkable findings.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

180 mg/kg bw/day

Basis for effect level:

other: maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 180 mg/kg/day.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The litter parameters (mean fetal weight and number of live fetuses) were comparable between the vehicle control group and rats treated up to the highest dose of 180 mg/kg/day, except at 45 mg/kg/day, the total number of fetuses and thus the mean litter size was significantly higher. This was considered incidental and toxicological not relevant as a similar trend was not observed at the higher doses.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The mean anogenital distance in male and female fetuses were comparable to the concurrent vehicle control, except at 90 mg/kg/day the mean anogenital distance of male fetuses was significantly higher and this finding was considered incidental as a similar trend was not observed at the highest dose of 180 mg/kg/day.

External malformations:

no effects observed

Description (incidence and severity):

No test item-related changes were observed during external observations of fetuses of rats treated up to 180 mg/kg/day.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no skeletal malformations observed in any litter at any of the tested dose leve.

Visceral malformations:

no effects observed

Description (incidence and severity):

No test item-related changes were observed during fresh visceral examination of fetuses of rats treated up to 180 mg/kg/day.

Fetal heads in all dose groups were normal when subjected to Wilsons Razor blade sectioning.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

180 mg/kg bw/day

Sex:

not specified

Basis for effect level:

other: fetal resorptions or post implantation loss were comparable to the control, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 180 mg/kg/d

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Treatment related:

no

Detailed results see attached background materials in "Overall remarks, attachements".

 

Summary of Maternal Survival, Pregnancy Status and Fetus Disposition

Parameters	Group No.	G1	G2	G3	G4
	Dose (mg/kg/day)	0	45	90	180
Total No. of rats found sperm positive / group		24	24	24	24
Duration of treatment		GD  5 to 19 (total 15 days)
Caesarean section (day of presumed gestation)		GD 20
Number of rats sacrificed at caesarean section		24	24	24	24
Number. of rats non-pregnant at caesarean section		2	1	1	5
Number of rats pregnant at caesarean section		22	23	23	19
Number of litters examined		22	23	23	19
Total number of fetuses		320	360	323	280
Total number of dead fetuses		0	0	0	0
Number of fetuses evaluated
a. External examination		320	360	323	280
b. Visceral examination		157	172	158	135
c. Skeletal examination		163	188	165	145

 

Summary of Clinical Signs and Mortality

Observation Type: All Types		Female
From Day 0 (Mating) to 20	G1 0 mg/kg/day	G2                G3 45                 90 mg/kg/day    mg/kg/day		G4 180 mg/kg/day
Normal	24	24	24	24
Killed - terminal kill	24	24	24	24

 

Summary of Maternal Body Weight of Pregnant Rats

Sex: Female Day(s) Relative to Mating			G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
Body	0 [a]	Mean	271.90	271.08	273.68	266.23
Weight (g)		SD N	21.61 22	21.10 23	21.55 23	22.09 19
		%Diff	-	-0.30	0.66	-2.08
	3 [a1]	Mean	289.51	288.27	291.64	284.12
		SD	19.20	22.19	21.92	22.74
		N	22	23	23	19
		%Diff	-	-0.43	0.74	-1.86
	5 [a1]	Mean	298.20	297.58	299.18	291.99
		SD	18.67	22.94	23.53	22.98
		N	22	23	23	19
		%Diff	-	-0.21	0.33	-2.08
	8 [a1]	Mean	309.26	308.05	309.07	300.71
		SD	19.11	24.47	25.32	22.66
		N	22	23	23	19
		%Diff	-	-0.39	-0.06	-2.76
	11 [a1]	Mean	327.25	325.27	326.06	318.19
		SD	20.58	24.66	26.88	22.94
		N	22	23	23	19
		%Diff	-	-0.60	-0.36	-2.77
	14 [a1]	Mean	343.17	341.58	342.62	336.86
		SD	22.09	26.01	28.16	25.64
		N	22	23	23	19
		%Diff	-	-0.46	-0.16	-1.84
	17 [a1]	Mean	377.16	376.82	374.26	371.34
		SD	25.80	26.65	28.71	25.66
		N	22	23	23	19
		%Diff	-	-0.09	-0.77	-1.54
	20 [a1]	Mean	425.27	428.12	418.19	421.92
		SD	33.79	30.87	35.93	31.82
		N	22	23	23	19
		%Diff	-	0.67	-1.66	-0.79

[a] - Anova & Dunnett(Log)

[a1] - Anova & Dunnett

 

Summary of Maternal Body Weight Gain of Pregnant Rats

Sex: Female Day(s) Relative to Mating			G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
Absolute	0 → 3 [a]	Mean	17.62	17.19	17.96	17.88
Weight Gain (g)		SD N	5.55 22	3.60 23	3.75 23	3.07 19
		%Diff	.	-2.41	1.95	1.50
	3 → 5 [a]	Mean	8.69	9.31	7.53	7.87
		SD	9.60	3.51	4.50	2.60
		N	22	23	23	19
		%Diff	.	7.09	-13.30	-9.39
	5 → 8 [a]	Mean	11.06	10.47	9.89	8.72
		SD	5.97	4.77	3.86	4.69
		N	22	23	23	19
		%Diff	.	-5.35	-10.56	-21.12
	8 → 11 [a]	Mean	17.98	17.23	16.99	17.48
		SD	7.12	3.09	3.93	4.34
		N	22	23	23	19
		%Diff	.	-4.21	-5.50	-2.81
	11 → 14 [a1]	Mean	15.93	16.31	16.56	18.67
		SD	5.04	4.61	3.50	4.85
		N	22	23	23	19
		%Diff	.	2.39	3.98	17.23
	14 → 17 [a]	Mean	33.99	35.24	31.63	34.48
		SD	9.34	7.38	5.32	6.74
		N	22	23	23	19
		%Diff	.	3.67	-6.93	1.44
	17 → 20 [a]	Mean	48.10	51.31	43.94	50.58
		SD	13.62	7.30	14.54	9.53
		N	22	23	23	19
		%Diff	.	6.66	-8.66	5.15

Sex: Female Day(s) Relative to Mating			G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
Absolute	0 → 5 [a]	Mean	26.31	26.50	25.50	25.76
Weight Gain (g)		SD N	11.57 22	5.67 23	4.70 23	4.15 19
		%Diff	.	0.73	-3.09	-2.10
	5 → 20 [a]	Mean	127.06	130.55	119.02	129.93
		SD	31.86	15.04	18.78	16.31
		N	22	23	23	19
		%Diff	.	2.74	-6.33	2.26
	0 → 20 [a]	Mean	153.37	157.05	144.51	155.69
		SD	31.39	16.04	19.75	17.45
		N	22	23	23	19
		%Diff	.	2.40	-5.78	1.51
Adjusted	GD20 - GU WT	Mean	339.32	337.46	335.55	333.03
Body weight (g)	[a]	SD N	28.02 22	24.20 23	28.54 23	22.92 19
		%Diff	.	-0.55	-1.11	-1.86
Adjusted	ADJ BWT-	Mean	41.12	39.88	36.38 *	41.04
Body weight Gain (g)	GD5BWT [a]	SD N %Diff	26.94 22 .	7.64 23 -3.02	13.28 23 -11.54	9.96 19 -0.20

[a] - Anova & Dunnett(Rank)
[a1] - Anova & Dunnett

 

Summary of Food Consumption of Pregnant Rats

Sex: Female Day(s) Relative to Mating			G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
Food Mean	0 → 3 [a]	Mean	23.66	23.56	23.38	22.37
Consumption (g/day)		SD N	2.08 22	2.45 23	2.46 23	2.15 19
		%Diff	.	-0.44	-1.22	-5.45
	3 → 5 [a]	Mean	25.02	25.50	24.16	24.22
		SD	2.76	2.63	2.87	2.14
		N	22	23	23	19
		%Diff	.	1.88	-3.47	-3.22
	5 → 8 [a]	Mean	26.02	25.47	24.65	23.78
		SD	2.57	3.16	3.07	2.02
		N	22	23	23	19
		%Diff	.	-2.14	-5.28	-8.63
	8 → 11 [a]	Mean	26.72	26.33	25.55	24.84
		SD	2.55	2.60	3.25	2.40
		N	22	23	23	19
		%Diff	.	-1.45	-4.37	-7.04
	11 → 14 [a]	Mean	27.87	27.89	27.03	27.43
		SD	3.06	2.66	3.41	2.57
		N	22	23	23	19
		%Diff	.	0.08	-3.01	-1.57
	14 → 17 [a]	Mean	28.45	28.43	27.50	27.39
		SD	3.52	2.44	2.76	2.17
		N	22	23	23	19
		%Diff	.	-0.08	-3.34	-3.73
	17 → 20 [a1]	Mean	30.00	29.16	27.27 *	29.30
		SD	3.66	1.85	3.80	3.09
		N	22	23	23	19
		%Diff	.	-2.78	-9.08	-2.32

 

Sex: Female Day(s) Relative to Mating			G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
Food Mean	0 → 5 [a]	Mean	24.21	24.33	23.69	23.11
Consumption (g/day)		SD N	1.99 22	2.30 23	2.44 23	1.99 19
		%Diff	.	0.52	-2.15	-4.53
	5 → 20 [a]	Mean	27.81	27.46	26.40	26.55
		SD	2.62	2.09	2.74	1.88
		N	22	23	23	19
		%Diff	.	-1.28	-5.07	-4.55
	0 → 20 [a]	Mean	26.91	26.68	25.72	25.69
		SD	2.27	2.08	2.61	1.76
		N	22	23	23	19
		%Diff	.	-0.87	-4.41	-4.54

[a] - Anova & Dunnett
[a1] - Anova & Dunnett(Rank): *: Statistically significant different from vehicle control at p < 0.05

 

 

Summary of Maternal Data

Sex: Female   Day(s) Relative to Mating			G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
Group size		N	24	24	24	24
Pregnant at C/S		N	22	23	23	19
Gravid Uterus Weight	[a]	Mean SD	85.942 11.480	90.667 13.931	82.640 17.631	88.896 15.576
Number of CorporaLutea	[a1]	Mean SD Sum	17.3 2.1 381	18.0 1.9 413	16.7 2.0 384	17.7 1.9 336
No. of Implantation	[a]	Mean SD Sum	16.0 1.9 353	16.3 2.4 374	15.0 2.8 346	15.7 2.5 298
Dams with Early Resorption		N	13	8	11	12
Number of Early Resorptions	[a]	Mean SD Sum	1.1 1.2 24	0.5 0.7 11	0.7 0.9 16	0.8 0.7 15
% Early Resorption /Animal	[a]	Mean SD	6.74 7.19	2.98 4.67	5.13 6.69	5.49 4.97
Dams with Late Resorption		N	6	3	6	2
Number of Late Resorptions	[a]	Mean SD Sum	0.4 0.8 9	0.1 0.3 3	0.3 0.6 7	0.2 0.5 3
% Late Resorption /Animal	[a]	Mean SD	2.46 5.00	0.71 1.89	1.86 3.46	0.90 2.79
Dams with Resorptions	[f]	N	17	9	14	12
Total Number of Resorption (Early + Late)	[f]	Mean SD Sum     Trend	1.5 1.2 33	0.6 0.9 14	1.0 1.0 23	0.9 1.0 18 0.7664
Pre-implantation Loss/Anima	[a]	Mean SD Sum	1.27 0.98 28	1.70 1.79 39	1.65 1.61 38	2.00 2.45 38
% Pre-implantation Loss	[a]	Mean SD	7.1 5.3	9.5 11.1	10.4 11.5	10.9 12.8
Post-implantation Loss/Animal	[a]	Mean SD Sum	1.50 1.22 33	0.61 0.89 14	1.00 1.00 23	0.95 0.97 18
% Post-implantation Loss (%)	[a]	Mean SD	9.2 7.5	3.7* 5.5	7.0 6.8	6.4 6.2

Note: Gross evaluation of placenta revealed no findings

 

[a] - Anova & Dunnett (Rank): *: Statistically significant different from vehicle control at p < 0.05

[a1] - Anova & Dunnett

[f] - Cochran Armitage, Chi-Squared & Fisher's Exact

 

• Summary of Litter Data

 

Sex: Female Day(s) Relative to Mating			G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
Total Number of fetuses	[a]	Sum	320	360*	323	280
Total Number of Dead Fetuses		Sum	0	0	0	0
Total Number of Live fetuses	[a]	Sum	320	360*	323	280
Mean Litter size	[a]	Mean SD N	14.5 1.9 22	15.7*  2.5 23	14.0 3.0 23	14.7 2.8 19
Live Male fetus		Sum	179	185	169	141
% Male Fetus			55.9	51.4	52.3	50.4
Mean Fetal Weight- Male (g)	[c]	Mean SD	3.873 0.212	3.873 0.245	3.902 0.300	4.044 0.468
Mean AGD- Male (mm)	[c1]	Mean SD	2.60 0.11	2.62 0.08	2.68* 0.12	2.64 0.10
Live Female fetus		Sum	141	175	154	139
% Female Fetus			44.1	48.6	47.7	49.6
Mean Fetal Weight- Female (g)	[c2]	Mean SD	3.663 0.193	3.684 0.202	3.730 0.277	3.825 0.460
Mean AGD- Female (mm)	[c3]	Mean SD	0.92 0.06	0.94 0.06	0.95 0.06	0.94 0.06
Mean Fetal Weight -Male+Female (g)	[c4]	Mean SD	3.785 0.189	3.777 0.217	3.809 0.267	3.940 0.446
Mean AGD Male + Female (mm)	[c5]	Mean SD	1.86 0.26	1.80 0.21	1.84 0.30	1.79 0.21

 

[a] - Anova & Dunnett(Rank):

[c] - Ancova/Anova & Dunnett (Rank); {Covariate(s): Number of Live Male Fetuses}

[c1] - Ancova/Anova & Dunnett; Covariate(s): Number of Live Male Fetuses}

[c2] - Ancova/Anova & Dunnett (Rank); {Covariate(s): Number of Live Female Fetuses}

[c3] - Ancova/Anova & Dunnett; Covariate(s): Number of Live Female Fetuses}

[c4] - Ancova/Anova & Dunnett (Rank); {Covariate(s): Number of Live Fetuses}

[c5] - Ancova/Anova & Dunnett; Covariate(s): Number of Live Fetuses}

 

   *: Statistically significant different from vehicle control at p < 0.05

 

Summary of Fetal External Observations

 

Exam Type: External	G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
Number of Live Fetuses Examined	320	360	323	280
Number of Litters Evaluated	22	23	23	19
Variants	None
Anamolies	None
Malformations	None

• Summary of Fetal Visceral Observations

Refer Appendix 9

 

Exam Type: Visceral	G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
Number of Live Fetuses Examined	157	172	158	135
Number of Litters Evaluated	22	23	23	19
Variants	None
Anamolies	None
Malformations	None

 

Summary of Fetal Skeletal Observations

Exam Type: Skeletal-Entire Number of Live Fetuses Examined: Number of Litters Evaluated:		G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
		163 22	188 23	165 23	145 19
Lumbar vertebrae
1st lumbar centrum, Split - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(5.3)
1st lumbar centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	2(1.2)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	2(8.7)	0(0.0)
8th lumbar centrum and arch, Extra - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	0(0.0)	0(0.0)
	Litters N(%)	0(0.0)	1(4.3)	0(0.0)	0(0.0)
thoracic vertebrae
13th thoracic centrum, Split - Anomaly	Fetuses N(%)	1(0.6)	1(0.5)	0(0.0)	1(0.7)
	Litters N(%)	1(4.5)	1(4.3)	0(0.0)	1(5.3)
13th thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	2(1.2)	5(2.7)	5(3.0)	0(0.0)
	Litters N(%)	2(9.1)	5(21.7)	5(21.7)	0(0.0)
13th thoracic centrum, Assymetrical dumbbell shaped - Anomaly	Fetuses N(%)	1(0.6)	0(0.0)	0(0.0)	0(0.0)
	Litters N(%)	1(4.5)	0(0.0)	0(0.0)	0(0.0)
13th thoracic centrum, Hypoplastic unilateral ossific - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(5.3)
12th thoracic centrum, Split - Anomaly	Fetuses N(%)	2(1.2)	3(1.6)	0(0.0)	0(0.0)
	Litters N(%)	2(9.1)	3(13.0)	0(0.0)	0(0.0)
12th thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	11(6.7)	24(12.8)	24(14.5)*	6(4.1)
	Litters N(%)	8(36.4)	12(52.2)	12(52.2)	5(26.3)
12th thoracic centrum, Assymetrical dumbbell shaped - Anomaly	Fetuses N(%)	1(0.6)	0(0.0)	0(0.0)	0(0.0)
	Litters N(%)	1(4.5)	0(0.0)	0(0.0)	0(0.0)
12th thoracic centrum, Asymmetrical split - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	1(0.6)	0(0.0)

 

Exam Type: Skeletal-Entire Number of Live Fetuses Examined: Number of Litters Evaluated:		G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
		163 22	188 23	165 23	145 19
thoracic vertebrae (Continued...)
12th thoracic centrum, Asymmetrical split - Anomaly	Litters N(%)	0(0.0)	1(4.3)	1(4.3)	0(0.0)
11th thoracic centrum, Split - Anomaly	Fetuses N(%)	2(1.2)	4(2.1)	0(0.0)	0(0.0)
	Litters N(%)	2(9.1)	3(13.0)	0(0.0)	0(0.0)
11th thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	15(9.2)	31(16.5)	29(17.6)*	14(9.7)
	Litters N(%)	11(50.0)	15(65.2)	17(73.9)	10(52.6)
11th thoracic centrum, Assymetrical dumbbell shaped - Anomaly	Fetuses N(%)	1(0.6)	0(0.0)	0(0.0)	0(0.0)
	Litters N(%)	1(4.5)	0(0.0)	0(0.0)	0(0.0)
10th thoracic centrum, Split - Anomaly	Fetuses N(%)	1(0.6)	2(1.1)	3(1.8)	0(0.0)
	Litters N(%)	1(4.5)	2(8.7)	2(8.7)	0(0.0)
10th thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	23(14.1)	35(18.6)	34(20.6)	14(9.7)
	Litters N(%)	12(54.5)	17(73.9)	15(65.2)	6(31.6)
9th thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	13(8.0)	16(8.5)	10(6.1)	6(4.1)
	Litters N(%)	9(40.9)	14(60.9)	6(26.1)	4(21.1)
8th thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	3(1.8)	6(3.2)	4(2.4)	3(2.1)
	Litters N(%)	3(13.6)	4(17.4)	4(17.4)	3(15.8)
7th thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	1(0.6)	0(0.0)	3(1.8)	1(0.7)
	Litters N(%)	1(4.5)	0(0.0)	2(8.7)	1(5.3)
6th thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	1(0.6)	0(0.0)	3(1.8)	0(0.0)
	Litters N(%)	1(4.5)	0(0.0)	2(8.7)	0(0.0)
5th thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	2(1.2)	1(0.5)	2(1.2)	0(0.0)
	Litters N(%)	2(9.1)	1(4.3)	2(8.7)	0(0.0)
4th thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	0(0.0)	1(0.7)

 

Exam Type: Skeletal-Entire Number of Live Fetuses Examined: Number of Litters Evaluated:		G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
		163 22	188 23	165 23	145 19
thoracic vertebrae (Continued...)
4th thoracic centrum, Dumbbell-shaped - Anomaly	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(5.3)
3rd thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	1(0.6)	0(0.0)	0(0.0)	0(0.0)
	Litters N(%)	1(4.5)	0(0.0)	0(0.0)	0(0.0)
2nd thoracic centrum, Dumbbell-shaped - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	1(0.6)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	0(0.0)
2nd thoracic centrum, Incomplete ossification - Variation	Fetuses N(%)	1(0.6)	0(0.0)	0(0.0)	1(0.7)
	Litters N(%)	1(4.5)	0(0.0)	0(0.0)	1(5.3)
1st thoracic centrum, Delayed skeletal ossification - Variation	Fetuses N(%)	1(0.6)	0(0.0)	0(0.0)	0(0.0)
	Litters N(%)	1(4.5)	0(0.0)	0(0.0)	0(0.0)
1st thoracic centrum, Incomplete ossification - Variation	Fetuses N(%)	1(0.6)	0(0.0)	1(0.6)	1(0.7)
	Litters N(%)	1(4.5)	0(0.0)	1(4.3)	1(5.3)
ribs
14th rib, Right, Rudimentary - Anomaly	Fetuses N(%)	10(6.1)	8(4.3)	8(4.8)	2(1.4)
	Litters N(%)	7(31.8)	7(30.4)	6(26.1)	2(10.5)
14th rib, Right, Accessory - Anomaly	Fetuses N(%)	3(1.8)	0(0.0)	0(0.0)	0(0.0)
	Litters N(%)	3(13.6)	0(0.0)	0(0.0)	0(0.0)
14th rib, Right, Extra - Anomaly	Fetuses N(%)	1(0.6)	0(0.0)	0(0.0)	0(0.0)
	Litters N(%)	1(4.5)	0(0.0)	0(0.0)	0(0.0)
14th rib, Left, Rudimentary - Anomaly	Fetuses N(%)	16(9.8)	16(8.5)	13(7.9)	5(3.4)
	Litters N(%)	11(50.0)	10(43.5)	8(34.8)	5(26.3)
14th rib, Left, Accessory - Anomaly	Fetuses N(%)	1(0.6)	0(0.0)	2(1.2)	1(0.7)
	Litters N(%)	1(4.5)	0(0.0)	2(8.7)	1(5.3)

 

Exam Type: Skeletal-Entire Number of Live Fetuses Examined: Number of Litters Evaluated:		G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
		163 22	188 23	165 23	145 19
ribs (Continued...)
14th rib, Left, Extra - Anomaly	Fetuses N(%)	1(0.6)	0(0.0)	0(0.0)	0(0.0)
	Litters N(%)	1(4.5)	0(0.0)	0(0.0)	0(0.0)
14th rib, Bilateral, Rudimentary - Anomaly	Fetuses N(%)	18(11.0)	11(5.9)	11(6.7)	15(10.3)
	Litters N(%)	11(50.0)	10(43.5)	8(34.8)	9(47.4)
14th rib, Bilateral, Accessory - Anomaly	Fetuses N(%)	3(1.8)	0(0.0)	1(0.6)	1(0.7)
	Litters N(%)	3(13.6)	0(0.0)	1(4.3)	1(5.3)
14th rib, Bilateral, Extra - Anomaly	Fetuses N(%)	0(0.0)	5(2.7)	1(0.6)	0(0.0)
	Litters N(%)	0(0.0)	1(4.3)	1(4.3)	0(0.0)
12th rib, Right, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(5.3)
12th rib, Right, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(5.3)
12th rib, Bilateral, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	2(1.2)	0(0.0)
	Litters N(%)	0(0.0)	1(4.3)	1(4.3)	0(0.0)
11th rib, Right, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	2(1.2)	1(0.7)
	Litters N(%)	0(0.0)	1(4.3)	2(8.7)	1(5.3)
11th rib, Right, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(5.3)
11th rib, Bilateral, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	1(0.6)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	0(0.0)
11th rib, Bilateral, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	2(1.2)	0(0.0)
	Litters N(%)	0(0.0)	1(4.3)	1(4.3)	0(0.0)

 

Exam Type: Skeletal-Entire Number of Live Fetuses Examined: Number of Litters Evaluated:		G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
		163 22	188 23	165 23	145 19
ribs (Continued...)
10th rib, Right, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	4(2.4)	2(1.4)
	Litters N(%)	0(0.0)	1(4.3)	4(17.4)	1(5.3)
10th rib, Right, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	1(4.3)	0(0.0)	1(5.3)
10th rib, Bilateral, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	1(0.6)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	0(0.0)
10th rib, Bilateral, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	2(1.2)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	0(0.0)
9th rib, Right, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	2(1.2)	2(1.4)
	Litters N(%)	0(0.0)	1(4.3)	2(8.7)	1(5.3)
9th rib, Right, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	1(4.3)	0(0.0)	1(5.3)
9th rib, Bilateral, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	2(1.2)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	0(0.0)
8th rib, Right, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	2(1.2)	2(1.4)
	Litters N(%)	0(0.0)	1(4.3)	2(8.7)	1(5.3)
8th rib, Right, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	1(4.3)	0(0.0)	1(5.3)
8th rib, Bilateral, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	2(1.2)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	0(0.0)
7th rib, Right, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	1(0.6)	3(2.1)
	Litters N(%)	0(0.0)	1(4.3)	1(4.3)	1(5.3)

 

Exam Type: Skeletal-Entire Number of Live Fetuses Examined: Number of Litters Evaluated:		G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
		163 22	188 23	165 23	145 19
ribs (Continued...)
7th rib, Right, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	1(4.3)	0(0.0)	1(5.3)
7th rib, Left, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(5.3)
7th rib, Bilateral, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	1(0.6)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	0(0.0)
7th rib, Bilateral, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	2(1.2)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	0(0.0)
6th rib, Left, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(5.3)
6th rib, Right, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	1(0.6)	3(2.1)
	Litters N(%)	0(0.0)	1(4.3)	1(4.3)	1(5.3)
6th rib, Right, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	1(4.3)	0(0.0)	1(5.3)
6th rib, Bilateral, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	1(0.6)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	0(0.0)
6th rib, Bilateral, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	2(1.2)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	0(0.0)
5th rib, Right, Wavy Rib, Slight - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	2(1.2)	2(1.4)
	Litters N(%)	0(0.0)	1(4.3)	2(8.7)	1(5.3)
5th rib, Right, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	0(0.0)	1(0.7)
	Litters N(%)	0(0.0)	1(4.3)	0(0.0)	1(5.3)

 

Exam Type: Skeletal-Entire Number of Live Fetuses Examined: Number of Litters Evaluated:		G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
		163 22	188 23	165 23	145 19
ribs (Continued...)
5th rib, Bilateral, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	2(1.2)	0(0.0)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	0(0.0)
4th rib, Right, Wavy Rib, Moderate - Anomaly	Fetuses N(%)	0(0.0)	0(0.0)	1(0.6)	1(0.7)
	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	1(5.3)
sternebrae
6th sternebra, Delayed skeletal ossification - Variation	Fetuses N(%)	0(0.0)	1(0.5)	1(0.6)	2(1.4)
	Litters N(%)	0(0.0)	1(4.3)	1(4.3)	2(10.5)
6th sternebra, Incomplete ossification - Variation	Fetuses N(%)	54(33.1)	51(27.1)	45(27.3)	42(29.0)
	Litters N(%)	20(90.9)	17(73.9)	16(69.6)	14(73.7)
5th sternebra, Hypoplastic - Anomaly	Fetuses N(%)	25(15.3)	23(12.2)	14(8.5)	17(11.7)
	Litters N(%)	17(77.3)	17(73.9)	13(56.5)	12(63.2)
5th sternebra, Asymmetrical ossification - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	0(0.0)	2(1.4)
	Litters N(%)	0(0.0)	1(4.3)	0(0.0)	2(10.5)
4th sternebra, Asymmetrical ossification - Anomaly	Fetuses N(%)	3(1.8)	3(1.6)	1(0.6)	4(2.8)
	Litters N(%)	3(13.6)	3(13.0)	1(4.3)	4(21.1)
4th sternebra, Incomplete ossification - Variation	Fetuses N(%)	4(2.5)	1(0.5)	3(1.8)	2(1.4)
	Litters N(%)	3(13.6)	1(4.3)	2(8.7)	2(10.5)
3rd sternebra, Hypoplastic - Anomaly	Fetuses N(%)	0(0.0)	1(0.5)	0(0.0)	0(0.0)
	Litters N(%)	0(0.0)	1(4.3)	0(0.0)	0(0.0)
3rd sternebra, Asymmetrical ossification - Anomaly	Fetuses N(%)	1(0.6)	2(1.1)	0(0.0)	1(0.7)
	Litters N(%)	1(4.5)	2(8.7)	0(0.0)	1(5.3)
3rd sternebra, Incomplete ossification - Variation	Fetuses N(%)	0(0.0)	0(0.0)	1(0.6)	1(0.7)

 

Exam Type: Skeletal-Entire Number of Live Fetuses Examined: Number of Litters Evaluated:		G1 0 mg/kg/day	G2 45 mg/kg/day	G3 90 mg/kg/day	G4 180 mg/kg/day
		163 22	188 23	165 23	145 19
sternebrae (Continued...)
3rd sternebra, Incomplete ossification - Variation	Litters N(%)	0(0.0)	0(0.0)	1(4.3)	1(5.3)
2nd sternebra, Hypoplastic - Anomaly	Fetuses N(%)	5(3.1)	5(2.7)	1(0.6)	4(2.8)
	Litters N(%)	3(13.6)	4(17.4)	1(4.3)	4(21.1)
2nd sternebra, Asymmetrical ossification - Anomaly	Fetuses N(%)	1(0.6)	0(0.0)	1(0.6)	0(0.0)
	Litters N(%)	1(4.5)	0(0.0)	1(4.3)	0(0.0)
skull
Supraoccipital, Incomplete ossification - Variation	Fetuses N(%)	14(8.6)	10(5.3)	5(3.0)*	17(11.7)
	Litters N(%)	6(27.3)	5(21.7)	4(17.4)	9(47.4)
Squamosal, Incomplete ossification - Variation	Fetuses N(%)	1(0.6)	0(0.0)	0(0.0)	2(1.4)
	Litters N(%)	1(4.5)	0(0.0)	0(0.0)	2(10.5)
Parietal, Incomplete ossification - Variation	Fetuses N(%)	21(12.9)	19(10.1)	12(7.3)	25(17.2)
	Litters N(%)	7(31.8)	12(52.2)	7(30.4)	11(57.9)
Interparietal, Incomplete ossification - Variation	Fetuses N(%)	17(10.4)	18(9.6)	8(4.8)	19(13.1)
	Litters N(%)	7(31.8)	9(39.1)	6(26.1)	10(52.6)
Hyoid, Incomplete ossification - Variation	Fetuses N(%)	10(6.1)	21(11.2)	14(8.5)	16(11.0)
	Litters N(%)	5(22.7)	11(47.8)	9(39.1)	9(47.4)

[Fetuses N] - Cochran Armitage, Chi-Squared & Fisher's Exact:
[Litters N] - Cochran Armitage, Chi-Squared & Fisher's Exact *: Statistically significant different from vehicle control at p < 0.05

 

 

 

Conclusions:

Based on the above findings, it is concluded that, No Observed Adverse- Effect Level (NOAEL) for

• Maternal toxicity is 180 mg/kg/day as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 180 mg/kg/day.

• Fetal developmental toxicity and Teratogenicity is 180 mg/kg/day as fetal resorptions or post implantation loss were comparable to the control, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 180 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the developmental toxicity of Genamin CH 020 in pregnant female Wistar rats and developing embryos/fetuses consequent to treatment of Genamin CH 020 in pregnant rats by oral gavage from gestation day (GD) 5 to 19. This study was intended to provide a rational basis for risk assessment in humans and to establish a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.

 

A total of 96,Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:

Group Nos.	Groups	Dose (mg/kg/day)	Dosage volume (mL/kg)	Concentration (mg/mL)	No. of Day 0 pregnant rats
G1	Vehicle control*	0	10	0	24
G2	Low dose	45	10	4.5	24
G3	Mid dose	90	10	9	24
G4	High dose	180	10	18	24

*Milli-Q^®Water

The following parameters and endpoints were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations].Approximately half the number of the fetuses from each litter were examined for visceral malformations and variations and the remaining half were evaluated for skeletal malformations and variations. In addition, from each dam, the thyroid was weighed and subjected to microscopic evaluation. Thyroid hormones levels were determined from the blood samples collected at terminal sacrifie (on GD 20).

 

Results of the study are summarized below:

 

·  Clinical signs and gross necropsy changes: There were no clinical signs, or mortalities in treated rats at any of the doses tested.

Grossly, at necropsy no abnormalities were detected.

·   Maternal Parameters: No treatment-related effects on maternal body weights and food consumption up to the highest tested dose of 180 mg/kg/day. The other maternal parameters comprising of uterine weight, implantations and early and late resorptions, post implantation loss were comparable to vehicle control group up to the high dose of 180 mg/kg/day. Gross evaluation of placenta revealed no remarkable findings.

·  Litter Parameters: No treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights, anogenital distance in male and female fetuses, were observed.

·  Fetal examination: External, visceral and skeletal examinations revealed no teratogenic effects attributed to the test item.

·  Thyroid hormone levels (T3, T4 and TSH), thyroid weights, and thyroid histology were unaffected by treatment.

 

Based on the above findings, it is concluded that, No Observed Adverse- Effect Level (NOAEL) for

 

• Maternal toxicity is 180 mg/kg/day as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 180 mg/kg/day.

 

• Fetal developmental toxicity and Teratogenicity is 180 mg/kg/day as fetal resorptions or post implantation loss were comparable to the control, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 180 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available (further information necessary)

Dose descriptor:

NOAEL

180 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

The reproduction and developmental toxicity potential of the registration substance was investigated in a reproduction/devlopmental toxicity screening study according to OECD 421 and a prenatal developmental toxicity study according to OECD 414. No adverse effects were observed regarding reproduction and developmental toxicity. Based on the available information, the submission substance does not have to be classified for reproduction and developmental toxicity in accordance with the criteria laid down in the EU Classification, Labellling and Packaging Regulation (1272/2008/EC).