Reaction mass of (Z)-3-(9-octadecenyloxy)propane-1,2-diol and alpha-(Z)-octadec-9-en-1-yl-omega-hydroxy-di[oxy[(hydroxymethyl)-1,2-ethanediyl]] and alpha-(Z)-octadec-9-en-1-yl-omega-hydroxy-tri[oxy[(hydroxymethyl)-1,2-ethanediyl]]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 May - 28 August 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted according to OECD Guideline 421 with deviations: temperature and humidity were not recorded for 4 days. Temperature and humidity were sometimes out of the target range. Only 7/10 pregnant females in the control group (and 6 delivered litters).

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

temperature and humidity not recorded for 4 days & sometimes out of target range. Only 7 pregnant females in contol group.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: 11-12 weeks
- Weight at study initiation: Males: 392-453 g (mean 420 g); Females: 268-302 g (mean 285 g)
- Housing: Animals were housed individually in wire-mesh cages (43.0 x 21.5 x 18.0 cm); Towards the end of gestation and during lactation, the females were individually housed, and with their litter after birth, in polycarbonate cages (43.0 x 21.5 x 20.0 cm) containing autoclaved wood shavings (SICSA, Alfortville, France) as nesting material.
- Diet: A04 C powdered maintenance diet (SAFE, Augy, France) distributed weekly, ad libitum
- Water: Tap water (filtered with a 0.22 μm filter), ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: Approximately 12 cycles/h of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

Route of administration:

oral: feed

Vehicle:

other: dietary admixture, in A04 C powdered maintenance diet

Details on exposure:

DIET PREPARATION
- For each concentration, a premix with the total amount of test item was prepared in the diet using a mixer. The final mix was prepared by dispersing the premix in the remaining diet in the mixer and then mixing for at least 10 minutes. Control animals were offered untreated diet alone.
- Storage temperature of food: Dietary admixtures were prepared on a weekly basis and were stored in closed bags at room temperature and protected from light prior to use.

STABILITY
- Satisfactory homogeneity and stability of dietary admixtures prepared at 500 or 20000 ppm was demonstrated during the study (Study No. 34827 AHS), over an 8-day storage period in open feeders and after 14 days of storage in closed bags at room temperature.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 1-14 days
- Proof of pregnancy: Presence of vaginal plug / sperm in vaginal lavage referred to as Day 0 post-coitum (p.c.).
- Pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Concentration of the test item in samples of each control and test item dietary admixtures prepared for use in Weeks 1, 4 and 8 was determined. On each day of analysis, duplicate samples was taken at three different levels (top, middle, bottom) of the mixer (groups 2, 3 and 4) or at a single level of the container (control group) for the determination of the test item concentration.
- Acceptance criterion: Measured concentration = nominal concentration ± 20 %.

Results: Test concentrations in the administered dietary admixtures analyzed remained within the acceptable range of -20 to -3 % of variation compared to the nominal values, except for highest dose group in Week 1 where the deviation from nominal value was -22 %.

Duration of treatment / exposure:

Males:
- 2 weeks before mating, during the mating (maximum of 2 weeks) and post-mating periods (at least 2 weeks) until sacrifice (at least 6 weeks in total).

Females:
- 2 weeks before mating, during the mating period (maximum of 2 weeks), during pregnancy and lactation, until Day 4 post-partum inclusive, (or until sacrifice for un-mated and non-pregnant females).

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
Males: 2175, 6525 and 14500 ppm; Females: 1950, 5850 and 13000 ppm [target dose levels 150, 450 and 1000 mg/kg bw/day]
Basis:
nominal in diet

No. of animals per sex per dose:

10

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of the results of 4-week toxicity study (Study No. 31261 TSR) in which the test item was administered by dietary admixture at the target dose-levels of 150, 450 and 1000 mg/kg bw/day.
- Rationale for animal assignment: During the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and/or clinical condition. They were then allocated to groups (by sex), using a stratification procedure based on body weight.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE AND CLINICAL OBSERVATIONS: Yes
Time schedule:
- Morbidity and mortality: Once a day during the acclimation period and at least twice a day during the treatment period.
- Clinical signs: Once daily

BODY WEIGHT: Yes
Time schedule for examinations:
- Male: Once before group allocation, on the first day of treatment (Day 1), then once a week until sacrifice.
- Female: Once before group allocation, on the first day of treatment (Day 1), then once a week until mated (or until sacrifice) and on Days 0, 7, 14 and 20 p.c. and Days 1 and 5 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Time schedule:
- Male: Once a week, over a 7-day period, from the first day of treatment until sacrifice.
- Female: Once a week, over a 7-day period, from the first day of treatment through gestation (Days 0-7, 7-14 and 14-20 p.c. intervals) and lactation (Days 1-5 post-partum interval) until sacrifice.
- During the mating period, the food consumption was noted for neither males nor females.
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Achieved intake of the test item (in terms of mg/kg bw/day) was calculated on a weekly basis for each test item-treated group as follows:
D = C x FC/BW; where, D = achieved dosage (mg/kg bw/day), C = nominal concentration of active test item (ppm: mg test item/10^6mg food), FC = mean food consumption (g/animal/day), BW = mean body weight (g)

PARTURITION:
- Females were allowed to litter normally and rear their progeny until Day 5 post-partum inclusive. Any sign of a difficult or prolonged parturition was recorded. The day of completed parturition was designated Day 1 post-partum. The length of gestation was calculated.

Oestrous cyclicity (parental animals):

Estrous cycle stage was recorded daily until the female was positive for mating.

Sperm parameters (parental animals):

Parameters examined in male animals:
- Testes and epididymis (total and cauda weight) weight were recorded.

Litter observations:

LITTER SIZE
- Total litter size and numbers of pups of each sex were recorded as soon as possible after birth.
- Litters were observed daily for recording of the number of live, dead and cannibalized pups.
- External examination of each pup was carried out and any external malformation was noted.

CLINICAL SIGNS
- Pups were observed daily for recording of the clinical signs or abnormal behavior.

BODY WEIGHT
- Weight of each pup was recorded on Days 1 and 5 post-partum.

Postmortem examinations (parental animals):

SACRIFICE: On completion of the treatment period, all animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.
- Males : All surviving animals were sacrificed after delivery of the majority of the females per group.
- Females: All surviving animals [on Day 5 post-partum, 24-26 days after the last day of the mating period for the females which did not mate, on or after Day 25 post-coitum for the females which had not delivered by Day 25 p.c.]

GROSS NECROPSY
- Macroscopic post-mortem examination of the external surfaces, all orifices, cranial cavity, external surfaces of the brain, thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.
- In all females, the number of implantation sites and corpora lutea was recorded.
- In the females which did not mate or were apparently non-pregnant, the presence of implantation sites on the uterine horns was checked using ammonium sulphide staining technique.

ORGAN WEIGTHS
- Epididymides (total and cauda weight), ovaries (with oviducts), prostate, seminal vesicles (with coagulating glands and their fluids) and testes were weighed.
- Ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

HISTOPATHOLOGY
- Microscopic examination was performed on the macroscopic lesions, ovaries, testes and epididymides (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) in all males and females of the control and high-dose groups.
- See table 7.8.1/1

Postmortem examinations (offspring):

SACRIFICE:
- Pups were sacrificed on Day 5 post-partum, by intraperitoneal injection of thiopental sodium.

GROSS NECROPSY
- Macroscopic examination was performed for all pups, including those found dead or prematurely sacrificed.
- Macroscopic lesions were preserved in 10 % buffered formalin (or another appropriate fixative) and were not microscopically examined in a first instance.

Statistics:

- Data other than organ weights: Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.
- Organ weights: PathData software (version 6.2b5) was used for the statistical analyses of organ weight data with a level of significance of 0.05 or 0.01.

Reproductive indices:

- Pre-implantation loss: [(Number of corpora lutea - Number of implantation sites) / Number of corpora lutea] X 100
- Post-implantation loss: [(Number of implantation sites - Number of live concepti) / Number of implantations] X 100
- Mating index: [Number of mated animals / Number of paired animals] X 100
- Fertility index: [Number of pregnant female partners / Number of mated pairs] X 100
- Gestation index: [Number of females with live born pups / Number of pregnant females] X 100

Offspring viability indices:

- Live birth index: [Number of live born pups / Number of delivered pups] X 100
- Viability index on Day 4 post-partum: [Number of surviving pups on Day 4 post-partum / Number of live born pups] X 100

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 performance:

no effects observed

MORTALITY (PARENTAL ANIMALS)
- No unscheduled deaths occurred during the study.

CLINICAL SIGNS (PARENTAL ANIMALS)
- No clinical signs were observed except the cutaneous lesion on the neck in one male at 450 mg/kg bw/day, however, this sign was not attributed to treatment with the test item.

BODY WEIGHT (PARENTAL ANIMALS)
- At 1000 mg/kg bw/day, lower body weight gain was observed in males and females during the first week of the pre-mating period (males: +28 g vs.+39 g; females: +13 g vs. +20 g) and the overall body weight gain of males from Day 1 to Day 43 was also lower than that of controls but not statistically significantly different. It was considered that these differences, which did not correlate with unaffected food consumption of this group, were no indicative of a test item treatment-related effect.
- During the gestation and lactation periods the body weight gain of test item-treated females was considered to be similar to that of controls.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Food consumption was unaffected by the treatment.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- Mean achieved dosages increased in a near dose-proportional manner and mean values were close to the targeted dose-levels of 150, 450 and 1000 mg/kg bw/day.
- See table 7.8.1/2.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- Estrous cycle was not affected by treatment with the test item at any dose level.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating data:
- No effect of treatment on mating parameters at any dose-level.

Fertility data:
- All the mated females were pregnant except 2/9 and 1/9 females at control and 450 mg/kg bw/day, respectively.
- One control female was sacrificed on Day 27 p.c. because of absence of delivery but had one implantation site at hysterectomy; all other pregnant females had live pups. The female fertility index was therefore considered not to have been affected by treatment with the test item as the incidence of non-pregnant females was not dose-related.

Delivery data:
- Mean duration of gestation, mean numbers of corpora lutea, implantations and pups born, as well as the pre- and post-implantation losses, were all comparable with the controls for all groups treated with the test item.
- Post-implantation loss was slightly higher at 1000 mg/kg bw/day when compared with controls (16 % vs. 12.9 %). This increase was attributed to one female which had a post-implantation loss of 56.3 % and was considered to be of no toxicological significance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Organ weight changes were considered not to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.
- Seminal vesicle weights for one high-dose male were low (1.2910 g and 0.22441 g respectively for absolute and relative weights versus 1.84 g and 0.32108 g respectively for control mean absolute and relative weights). These low weights correlated with the reduced size of the left vesicle noted at necropsy and with decreased glandular content at microscopic examination. Since this finding was unilateral and isolated, it was considered to be incidental and unrelated to the test item.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- All macroscopic findings were those commonly recorded in the Sprague-Dawley rats and none were considered to be related to the test item administration.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- All microscopic findings noted in treated animals were considered to be incidental changes as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity and/or are common background findings for the species.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No mortality, no clinical signs, no effects on body weight or food consumption and no adverse effects on reproductive performance were observed at any of the doses tested.

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

VIABILITY AND CLINICAL SIGNS (OFFSPRING)
- Pup mortality was higher at 450 and 1000 mg/kg bw/day (9.5 and 17.6 %, respectively, vs. 0 % in controls) when compared to the controls. In the high-dose group, the entire litter of one dam was died and ante-mortem signs of paleness and/or coldness to the touch were noted. Pup mortality and clinical signs recorded at 450 and 1000 mg/kg bw/day were treatment-related.
- At 150 mg/kg bw/day, no clinical signs were observed and the mortality of pups was very low and considered not to be related to the test item treatment.

BODY WEIGHT (OFFSPRING)
- Body weight gain of male and female pups from the test item-treated groups was similar to that of the control group.

SEX RATIO (OFFSPRING)
- Percentage of male pups ranged between 42.6 and 56.2 % on Days 1 and 5 post-partum which was considered to be within the normal range.

GROSS PATHOLOGY (OFFSPRING)
- No findings were observed in the pups sacrificed on Day 5 post-partum.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Reproductive effects observed:

not specified

Table 7.8.1/2: Achieved dosages

Mean achieved dosages (mg/kg bw/day) in males
Concentration (ppm)	2175	6525	14500
Pre-mating (Days 1-15)	141	388	938
Comparison with target dose-levels (%)	-6	-14	-6
Mean achieved dosages (mg/kg bw/day) in females
Concentration (ppm)	1950	5850	13000
Pre-mating (Days 1-15)	141	450	1094
Gestation (GD 0-20)	143	473	1044
Lactation (Days 1-5 p.p.)	208	511	1432
Overall mean (females)	164	478	1190
Comparison with target dose-levels (%)	+9	+6	+19

 

GD: Gestation Day, p.p.: post-partum

Table 7.8.1/3: Reproductive data

Dose-level (mg/kg bw/day)	0	150	450	1000
Summary of mating data
Paired males + females	10 + 10	10 + 10	10 + 10	10 + 10
Mating index (%)	90	80	90	100
Mean pre-coital time (days)	3.8	4.9	3.6	3.3
Fertility data
Fertility index (%)	77.8	100	88.9	100
Gestation index (%)	85.7	100	100	100
Delivery data
Litters with live born pups	6	8	8	10
Duration of gestation (days)	21.8	22.0	21.9	22.1
Mean number of corpora lutea	18.7	16.9	17.3	17.5
Mean number of implantations	17.0	16.3	16.3	16.2
Mean pre-implantation loss (%)	7.5	3.5	5.7	6.9
Mean number of pups delivered	14.8	14.8	14.5	13.6
Live birth index (%)	100	100	100	100
Mean post-implantation loss (%) (calculated manually)	12.9	9.2	11.0	16.0

 

Table 7.8.1/4: Pup mortality and clinical signs

Dose-level (mg/kg bw/day)	0	150	450	1000
Pup mortality
between Days 1 and 4 p.p.	0 (0 %)	3 (2.5 %)	11** (9.5 %)	24 # (17.6 %)
Viability index on day 4 p.p.(%)	100.0	97.5	90.5	82.4
litters affected	0/6 (0 %)	2/8 (25 %)	4/8 (50 %)	6/10 (60 %)
Clinical signs
coldness to touch^a	0/2	0/0	2/2	11/13
paleness^a	0/2	0/0	2/2	2/13
Total litters affected by clinical signs	0/6 (0 %)	0/8 (0 %)	1/8 (13 %)	3/10 (30 %)

 

p.p.: post-partum, **: p<0.01; #: p<0.001, a: number of pups affected compared with the total of pups showing signs.

Conclusions:

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of CHIMEXANE NB was considered to be 1000 mg/kg bw/day (concentration of 14500 ppm for the males or 13000 ppm for the females).
At 450 and 1000 mg/kg bw/day, the viability index on Day 4 p.p was lower than that of controls (90.5 and 82.4 %, respectively, vs.100.0 % in controls; statistically significant but within the historical range provided by the laboratory - 80.3 -100%). Data from this study, escpecialy the pups mortality reported at top dose, were reviewed by an Expert and compared to historical control data provided by the laboratory. The lower viability observed at the high dose was considered within the scope of biological variability of this species and strain.

Therefore, for embryo-fetal / pup developmental toxicity up to day 5 post-partum, the No Observed Adverse Effect Level (NOAEL) was considered to be at 1000 mg/kg/day.

Executive summary of the Expert Report

• Data from Reproduction/Developmental Toxicity Screening Test on Chimexane NB were reviewed and compared to contemporaneous control data (from a similar screening study) and historical control data provided by the CRO
• Control data in this screening study is not representative of expected (historical control) values as the small sample size determined by the study design (OECD 421) has been further reduced by one non-mated female, two non-pregnant and one total pre-partum litter loss, resulting in only 6 litters for evaluation, reducing the power of the study to detect adverse effects in treated groups. Data from the treated groups has therefore been compared to contemporaneous control data and a broad range of historical control values which provide insight into the biological variation expected in this species and strain
• Data for the high dose group include one animal which had total litter loss by day 2 post-partum. This is known to occur spontaneously and has been observed in a control animal in one of the 11 studies provided as historical data. There is no data to suggest that this animal was unduly sensitive to the test article and as the only recorded effect on pups was ‘cold to touch’ the deaths were considered secondary to poor maternal care
• As no other parameters on this study showed any treatment-related effects and as there were no effects indicating a potential mode of action observed in other toxicity studies with Chimexane NB (Prenatal Developmental Toxicity, 90-day Repeat Dose Toxicity, Genotoxicity), it was concluded that this effect was unlikely to represent reproductive or developmental toxicity
• Toxicity studies (Reproduction/Developmental Toxicity Screening Test, Prenatal Developmental Toxicity Study, 90-day Repeat Dose Toxicity Study) with the structurally similar compound Chimexane NC also show no adverse effects on reproduction or development and are considered to contribute to the weight of evidence for no effect on reproductive or developmental toxicity
• The lower viability observed in the high dose group of the Chimexane NB Reproduction/Developmental Toxicity Screening Test was considered, within the limitations of the study design, to be within the scope of biological variability of this species and strain rather than a definitive effect on reproductive function or developmental parameters, and this isolated finding is therefore unlikely to be a cause for concern

Executive summary:

In a reproduction/developmental toxicity screening study performed according to OECD Guideline 421 and in compliance with GLP, CHIMEXANE NB was administered by dietary admixture to groups (10/sex/dose) of Sprague-Dawley, Rj Han: SD rats at the dose levels of 0, 2175, 6525 and 14500 ppm for the males and 0, 1950, 5850 and 13000 ppm for the females (equivalent to target dose-levels of 0, 150, 450 and 1000 mg/kg bw/day) for 15 days before mating, through the mating and post-mating periods until sacrifice for the males, and through gestation and the beginning of the lactation period (until Day 4 post-partum (p.p.) inclusive) for the females. Clinical signs and mortality were checked daily. Body weight and food consumption were recorded at designated intervals throughout the study, except during the mating period when the food intake was not recorded. After 15 days of dosing, the males and females were paired and the females were allowed to litter normally and rear their progeny until Day 5 p.p. Males were sacrificed approximately 2 weeks after the end of the mating period, females on Day 5 p.p. The animals were sacrificed and subjected to a complete macroscopic examination and designated organs were weighed and examined microscopically. The pups were observed daily for clinical signs, sexed and weighed on Days 1 and 5 p.p and sacrificed on Day 5 p.p., and subjected for macroscopic examination.

 

No mortality and no clinical signs attributed to treatment with the test item were observed. No effects on body weight or food consumption were observed. The reproductive performance of males and females, mating, fertility, gestation and live birth indices were similar to that of the control group. No treatment-related effects were noted on testis or epididymis weights and no treatment-related findings were recorded at necropsy or microscopic examination of the testes, epididymides or ovaries in treated rats. There were no treatment-related effects on pup mortality, clinical signs or body weight at 150 mg/kg bw/day. At 450 and 1000 mg/kg bw/day, the viability index on Day 4 p.p was lower than that of controls (90.5 and 82.4 %, respectively, vs.100.0 % in controls; statistically significant but within the historical range provided by the laboratory - 80.3 -100%). No macroscopic findings were observed in the pups sacrificed on Day 5 post-partum.

Data from this study, escpecialy the pups mortality reported at top dose, were reviewed by an Expert and compared to historical control data provided by the laboratory. The lower viability observed at the high dose was considered within the scope of biological variability of this species and strain. Therefore, under the test conditions, the No Observed Adverse Effect Level (NOAEL) of CHIMEXANE NB was considered to be 1000 mg/kg bw/day (concentration of 14500 ppm for the males or 13000 ppm for the females) and 1000 mg/kg bw/day (overall mean achieved dosage 1190 mg/kg/day) for embryo-fetal / pup developmental toxicity up to day 5 post-partum, respectively in Sprague-Dawley rats

Executive summary of the Expert Report •Data from Reproduction/Developmental Toxicity Screening Test on Chimexane NB were reviewed and compared to contemporaneous control data (from a similar screening study) and historical control data provided by the CRO •Control data in this screening study is not representative of expected (historical control) values as the small sample size determined by the study design (OECD 421) has been further reduced by one non-mated female, two non-pregnant and one total pre-partum litter loss, resulting in only 6 litters for evaluation, reducing the power of the study to detect adverse effects in treated groups. Data from the treated groups has therefore been compared to contemporaneous control data and a broad range of historical control values which provide insight into the biological variation expected in this species and strain •Data for the high dose group include one animal which had total litter loss by day 2 post-partum. This is known to occur spontaneously and has been observed in a control animal in one of the 11 studies provided as historical data. There is no data to suggest that this animal was unduly sensitive to the test article and as the only recorded effect on pups was ‘cold to touch’ the deaths were considered secondary to poor maternal care •As no other parameters on this study showed any treatment-related effects and as there were no effects indicating a potential mode of action observed in other toxicity studies with Chimexane NB (Prenatal Developmental Toxicity, 90-day Repeat Dose Toxicity, Genotoxicity), it was concluded that this effect was unlikely to represent reproductive or developmental toxicity •Toxicity studies (Reproduction/Developmental Toxicity Screening Test, Prenatal Developmental Toxicity Study, 90-day Repeat Dose Toxicity Study) with the structurally similar compound Chimexane NC also show no adverse effects on reproduction or development and are considered to contribute to the weight of evidence for no effect on reproductive or developmental toxicity •The lower viability observed in the high dose group of the Chimexane NB Reproduction/Developmental Toxicity Screening Test was considered, within the limitations of the study design, to be within the scope of biological variability of this species and strain rather than a definitive effect on reproductive function or developmental parameters, and this isolated finding is therefore unlikely to be a cause for concern

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Appropriate database

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a reproduction/developmental toxicity screening study performed according to OECD Guideline 421 and in compliance with GLP, CHIMEXANE NB was administered by dietary admixture to groups (10/sex/dose) of Sprague-Dawley, Rj Han: SD rats at the dose levels of 0, 2175, 6525 and 14500 ppm for the males and 0, 1950, 5850 and 13000 ppm for the females(equivalent to target dose-levels of 0, 150, 450 and 1000 mg/kg bw/day) for 15 days before mating, through the mating and post-mating periods until sacrifice for the males, and through gestation and the beginning of the lactation period (until Day 4post-partum (p.p.)inclusive) for the females.

The reproductive performance of males and females, mating, fertility, gestation and live birth indices were similar to that of the control group. No treatment-related effects were noted on testis or epididymis weights and no treatment-related findings were recorded at necropsy or microscopic examination of the testes, epididymides or ovaries in treated rats.

the NOAEL for systemic toxicity was 1000 mg/kg bw/day and the NOEL for mating and fertility was 1000 mg/kg bw/day.

Short description of key information:
In a GLP study conducted according to OECD guideline 421, the NOAEL for systemic toxicity was 1000 mg/kg bw/day and the NOEL for mating and fertility was 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

In a GLP study conducted according to OECD guideline 414, the NOAEL for maternal toxicity was 450 mg/kg bw/day (nominal concentration) and the NOAEL for developmental toxicity was 1000 mg/kg bw/day. In a GLP study conducted according to OECD guideline 421, the dose-level of 1000 mg/kg/day was also considered to be the NOAEL for developmental toxicity in pups at day 5 post-partum.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 May 2008 - 22 April 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted according to OECD Guideline 414 with deviations: acclimation period followed for 4 or 5 days instead of 5 days; temperature and relative humidity were not recorded for one day; single instead of duplicate analyses was performed for determination of content in control group on Day 1

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

acclimation period followed for 4 or 5 days instead of 5 days; temperature and relative humidity were not recorded for one day; single instead of duplicate analyses was performed for determination of content in control group on Day 1

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 267 g (mean body weight)
- Housing: Animals were housed individually in wire-mesh cages (43.0 x 21.5 x 18.0 cm).
- Diet: A04 C powdered maintenance diet (SAFE, Augy, France) distributed weekly, ad libitum
- Water: Tap water (filtered with a 0.22 μm filter), ad libitum
- Acclimation period: 4 or 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: About 12 cycles/h of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

Route of administration:

oral: feed

Vehicle:

other: dietary admixture, in A04 C powder maintenance diet

Details on exposure:

DIET PREPARATION
- For each concentration, a premix with the total amount of test item was prepared in the diet using a mixer. The final mix was prepared by dispersing the premix in the remaining diet in the mixer and then mixing for at least 10 minutes.
- Storage temperature of food: Dietary admixtures were prepared on a weekly basis and were stored in closed bags at room temperature and protected from light prior to use.

STABILITY
- Satisfactory homogeneity and stability of dietary admixtures prepared at 500 or 20000 ppm was previously demonstrated during the study (Study No. 34827 AHS), over a 14-day storage period in open feeders and in closed bags at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Concentration of the test item was determined in samples of each control and test item dietary admixture prepared for use on the first day of treatment of the first female and on the last day of treatment of the last female.
- Acceptance criterion: Measured concentration = nominal concentration ± 20 %.

Results: Test item concentrations in the administered dietary admixtures analyzed on the first day of treatment of the first female and on the last day of treatment of the last female remained within an acceptable range of [- 17 % to + 4 %] of variation compared to the nominal values.

Details on mating procedure:

- Impregnation procedure: Purchased timed pregnant; the females were mated at the breeder's facilities (Janvier, Le Genest-Saint-Isle, France).
- Proof of pregnancy: Detection of vaginal plug referred to as Day 0 post-coitum (p.c.).

Duration of treatment / exposure:

15 days (Days 6-20 p.c.)

Frequency of treatment:

Once daily; animals were allowed the dietary admixture ad libitum

Duration of test:

22 days (Days 0-21 p.c.)

No. of animals per sex per dose:

24 mated females/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of the results of 4-week toxicity study (Study No. 31261 TSR) in which the test item was administered by dietary admixture at the target dose-levels of 150, 450 and 1000 mg/kg bw/day.
- Rationale for animal assignment: Before the beginning of the treatment period, the animals were allocated to the groups, according to a stratified procedure based on body weight recorded on Day 2 p.c., to ensure comparatively similar mean body weights among groups.

Maternal examinations:

CAGE SIDE & CLINICAL OBSERVATIONS: Yes
Time schedule:
- Mortality or signs of morbidity: Once a day before the start of treatment and then twice a day during treatment period, including weekends and public holidays.
- Clinical signs: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Achieved intake of the test item was calculated for the following intervals 6-9, 9-12, 12-15, 15-18 and 18-21 p.c. for each test item-treated group as follows:
D = C x FC/BW; where, D = achieved dosage (mg/kg bw/day), C = nominal concentration of active test item (ppm: mg test item/10^6 mg food), FC = mean food consumption (g/animal/day), BW = mean body weight (g)

POST-MORTEM EXAMINATIONS: Yes
- Time schedule: On Day 21 p.c., females were sacrificed by inhalation of carbon dioxide followed by cervical dislocation and were subjected to a macroscopic post-mortem examination (principal thoracic and abominal organs).

OTHER:
Preservation of tissues:
- Any macroscopic lesions observed were sampled and kept preserved in 10 % buffered formalin.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number and distribution of implantations: Yes
- Number and distribution of early and late resorptions: Yes
- Number and distribution of uterine scars: Yes
- Number and distribution of dead and live fetuses: Yes
- Gross evaluation of placentas: Yes

Fetal examinations:

EXAMINATION OF FETUSES:
The first 20 litters/group were examined as follow:
- Live fetuses were sacrificed by a subcutaneous injection of thiopental sodium.
- Body weight of fetuses: Body weight of each live fetus was recorded.
- Sex of fetuses: Sex of each fetus [excluding the autolyzed fetus] was determined at the time of hysterectomy by visual assessment of the anogenital distance and was confirmed by examination of the sexual organs at the time of detailed dissection of the soft tissues or at evisceration.
- Dead fetuses: Dead fetus was fixed in Harison’s fluid for a possible further examination.
- External examinations: Yes [all per litter excluding the autolyzed fetus]; each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.
- Soft tissue examinations: Yes, approximately half of the live fetuses in each litter were subjected to a detailed dissection of the soft tissues, which included observation of all the organs and structures of the neck, thorax and abdomen. The fetuses were then eviscerated and were fixed in Harison’s fluid for examination of the structures of the head.
- Skeletal examinations: Yes [all the remaining litters]; eviscerated and fixed with ethyl alcohol, stained with alizarin red S and alcian blue and examined for skeletal abnormalities including the observation of all the bones and cartilage structures of the head, spine, rib cage, pelvis and limbs.

For the remaining litters, fetuses were fixed with ethyl alcohol, stained with alizarin red S and alcian blue for further possible examination.

Statistics:

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

Indices:

- Pre-implantation loss: [(Number of corpora lutea - Number of implantation sites) / Number of corpora lutea] X 100
- Post-implantation loss: [(Number of implantation sites - Number of live fetuses) / Number of implantation sites] X 100
- Fetal or litter incidence: (Total number of fetuses or litters with a particular finding / Total number of fetuses or litters examined)X 100
- Mean proportion of affected fetuses: (Sum of proportion of fetuses affected in each litter / Total number of litters examined) X 100

Historical control data:

Historical background data was used to compare the incidences of developmental toxicity.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Pregnancy status:
- All mated animals were pregnant, except 2/24 control females and 1/24 test-item treated females per group, and had live fetuses at term.

Mortality and clinical signs:
- No mortality was observed.
- No clinical signs were observed except cutaneous lesions on the head in one female and misshapen ear in another female were observed at 150 mg/kg bw/day, however these two events were considered fortuitous in origin and unrelated to treatment with the test item.

Body weight and body weight change:
- Lower body weight gain was observed during the first 3 days of treatment in females receiving 1000 mg/kg bw/day when compared to controls (13 g vs. 23 g) and this effect was transient and coincided with the lower food consumption. However, the terminal body weight recorded on Gestation Day (GD) 21 was not statistically significantly different from the control value and the net body weight and the carcass weights did not differ from that of the control group. It could be concluded that the test item affected the body weight gain transiently.
- At 150 and 450 mg/kg bw/day, the body weight gain and the body weight were unaffected by the treatment with the test item.

Food consumption:
- Mean food consumption of females treated at 1000 mg/kg bw/day was lower than controls [21 g vs. 26 g from GD 6 to GD 9 (p<0.001), 24 g vs. 26 g from GD 9 to GD 12 (p<0.05) and 26 g vs. 29 g from GD 12 to GD 15 (p<0.05)] and they were considered to be test item treatment-related.
- At 150 and 450 mg/kg bw/day, the food consumption was considered to have been unaffected by the test item treatment.

Maternal necropsy findings:
- There were no necropsy findings which were attributable to the test item treatment. The single observation recorded concerned one control female with dilated and presence of serous content in the uterus; this animal was not pregnant.

Gravid uterus weight:
- Mean gravid uterus weight of test item-treated females was similar to that of the control group.

Litter data:
- Number of resorptions was similar between the test item-treated groups and the controls.
- No dead fetuses were noticed except one dead fetus at 450 mg/kg bw/day.
- No differences in the mean number of implantation sites and the number of live fetuses between the test item-treated and control groups.
- Sex ratio and mean fetal body weight were similar in the control and the treated groups.

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
External observations:
- Total of 236, 249, 257 and 231 fetuses from the control, 150, 450 and 1000 mg/kg bw/day groups were examined. Apart from one fetus of the control group with omphalocele and a dead fetus at 450 mg/kg bw/day which was autolyzed, there were no remarkable external findings in any of the fetuses examined.

Soft tissue observations:
- Total of 112, 118, 123 and 110 fetuses from the control, 150, 450 and 1000 mg/kg bw/day groups were examined.
- Fetal soft tissue variations and malformations observed were randomly distributed within the groups including the control. Furthermore, as the incidence of the observations was very low and/or incidences of variations were within historical background data (dilated renal pelvis or dilated ureter, short or absent innominate artery), they were considered to be unrelated to treatment.

Skeletal and cartilage observations:
- Total of 124, 131, 134 and 121 fetuses from the control, 150, 450 and 1000 mg/kg bw/day groups were examined.
- As the incidences of skeletal and cartilage variations and malformations were low, recorded with similar frequency among the control and treated groups and in the absence of statistical differences, they were considered to be unrelated to treatment.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 7.8.2/1: Mean achieved dosages (mg/kg bw/day) between gestation Days 6 and 21

Concentration (ppm)	1878	5634	12519
GD 6 to 21	159	464	992
Target dose-levels (mg/kg bw/day)	150	450	1000
(% from target dose-level)	+ 6 %	+ 3 %	- 1 %

 

GD: gestation day

Mean achieved dosages increased in a nearly dose-proportional manner and mean values were very close to the targeted dose-levels of 150, 450 and 1000 mg/kg bw/day.

Table 7.8.2/2: Body weight and body weight change (g)

Dose-level (mg/kg bw/day)	0	150	450	1000
BW, GD 6	269	267	265	264
BW, GD 21	418	421	414	404
BWG, GD 6-9	23	20	22	13 #
BWG, GD 6-21	+149	+154	+149	+139
Carcass weight	324.8	323.4	314.3	312.3
Net weight change from GD 6	+55.9	+56.6	+49.4	+48.0

 

GD: gestation day, BW: body weight, BWG: body weight gain, #: p<0.001.

 

Table 7.8.2/3: Food consumption

Dose-level (mg/kg bw/day)		0	150	450	1000
Days 2-6	Mean	23 d	22	23	22
	SD	2	3	3	2
Days 6-9	Mean	26 d	25	24	21 #
	SD	3	3	3	4
Days 9-12	Mean	26 d	27	27	24*
	SD	4	3	3	4
Days 12-15	Mean	29 d	29	28	26*
	SD	3	3	2	3
Days 15-18	Mean	30 d	30	30	28
	SD	3	4	2	4
Days 18-21	Mean	28 d	28	26	27
	SD	3	5	5	3

 

No. of animals: 22 for control and 23 for test item treated groups, mean: mean values/grams per day, d=ANOVA + Dunnett-test, * = p<0.05, # = p<0.001

Conclusions:

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of CHIMEXANE NB was considered to be 5634 ppm (equivalent to 450 mg/kg bw/day) for maternal toxicity and the No Observed Effect Level (NOEL) of CHIMEXANE NB was considered to be 12519 ppm (equivalent to 1000 mg/kg bw/day) for developmental toxicity in Sprague-Dawley rats.

Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, CHIMEXANE NB was administered by dietary admixture to groups of mated female Sprague-Dawley, Rj Han: SD rats (24/dose) at the dose levels of 0, 1878, 5634 and 12519 ppm (equivalent to target dose-levels of 0, 150, 450 and 1000 mg/kg bw/day) from Days 6 to 20 post-coitum. Clinical signs and mortality were checked daily and maternal body weight and food consumption were recorded at designated intervals. On Day 21 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The gravid uterine weight, numbers of implantations, uterine scars, live and dead fetuses, early and late resorptions and corpora lutea were recorded. Fetuses were sexed, weighed and examined for external, soft tissue and skeletal malformations.

All mated animals were pregnant except 2/24 control females and 1/24 test item-treated females per group. No deaths and no clinical signs related to treatment with the test item were recorded. The body weight gain and food consumption of animals at 1000 mg/kg bw/day were transiently lower than controls. However, the terminal body weight was not statistically significantly different from controls. None of the litter parameters evaluated were affected and there were no fetal external findings with treatment-relationship in any of the fetuses examined. No findings at visceral or skeletal (including cartilage) fetal examinations were considered to be related to the test item at the concentrations tested.

 

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of CHIMEXANE NB was considered to be 5634 ppm (equivalent to 450 mg/kg bw/day) for maternal toxicity and the No Observed Effect Level (NOEL) of CHIMEXANE NB was considered to be 12519 ppm (equivalent to 1000 mg/kg bw/day) for developmental toxicity in Sprague-Dawley rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

450 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

Additional information

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, CHIMEXANE NB was administered by dietary admixture to groups of mated female Sprague-Dawley,Rj Han: SDrats (24/dose) at the dose levels of 0, 1878, 5634 and 12519 ppm (equivalent to target dose-levels of 0, 150, 450 and 1000 mg/kg bw/day) from Days 6 to 20 post-coitum.

Transient reductions of the body weight gain and food consumption were recorded in dams given 1000 mg/kg/day, without affecting the terminal body weight. No other maternal findings were observed during the study. None of the litter parameters recorded (implantations, live fetuses, percentage of male/female fetuses, fetal body weight) were affected. There were no indications of any treatment-related variations or malformations in fetuses.

Consequently, under the experimental conditions of this study, the dose-level of 450 mg/kg/day was consecutively considered to be the No Observed Adverse Effect Level (NOAEL) for maternal toxicity, and 1000 mg/kg/day was considered to be the No Observed Effect Level (NOEL) for developmental toxicity.

In a reproduction/developmental toxicity screening study performed according to OECD Guideline 421 and in compliance with GLP, CHIMEXANE NB was administered by dietary admixture to groups (10/sex/dose) of Sprague-Dawley, Rj Han: SD rats at the dose levels of 0,2175, 6525 and 14500 ppm for the males and 0, 1950, 5850 and 13000 ppm for the females(equivalent to target dose-levels of 0, 150, 450 and 1000 mg/kg bw/day) for 15 days before mating, through the mating and post-mating periods until sacrifice for the males, and through gestation and the beginning of the lactation period (until Day 4post-partum (p.p.)inclusive) for the females.

At 450 and 1000 mg/kg bw/day, the viability index on Day 4 p.p was lower than that of controls (90.5 and 82.4 %, respectively,vs.100.0 % in controls; statistically significant), but these values were within the historical control range (80.3 to 100%), and were considered to be not treatment related. No macroscopic findings were observed in the pups sacrificed on Day 5 post-partum.Therefore, The dose-level of 1000 mg/kg/day was considered to be the NOAEL for embryo-fetal and pup developmental toxicity up to day 5 post-partum.

Taken together, the overall NOAEL for pup developmental toxicity was considered at 1000 mg/kg/day.