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Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 March 1996 to 22 December 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed to GLP and in line with the standardised guidelines OECD 416 and EPA OPP 83-4, with no deviations thought to impact the reliability of the presented results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries 59 NohSan No. 4200, "Reproduction Study" January 28 1985
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: European Communities Commission Directive 87/302/EEC, "Two-Generation Reproduction Toxicity Test", OJ No L133/47, November 18, 1987
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Tif: RAI f, hybrids of RII/1 x RII/2
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P) 6 - 7 weeks
- Weight at study initiation (mean): (P) Males: 218.5 - 219.9 g; Females: 160.8 - 163.9 g
- Housing: Individually
- Diet: pelleted, certified standard feed ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): about 16 air changer per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION:
- Rate of preparation of diet : On average every 4 weeks
- Mixing appropriate amounts with: Ground feed pellets were mixed with the appropriate quantity of test material, hydrated and repressed to pellets
- Storage temperature of food: The diet was stored in un-opened sacks or in polyurethane of stainless steel bins with dust-tight lids, at room temperature.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 19 days
- Proof of pregnancy: presence of vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Samples: The stability of the test substance in feed mixtures was assessed after 6 weeks storage and content under actual conditions of administration, test substance content, homogeneity and stability were measured in the pellet samples.
- Sample storage: Before analysis, samples were frozen at approximately – 20 °C
- Preparation of samples: Depending on the nominal values, aliquots of the extracts were either directly diluted with the mobile phase or evaporated to dryness and the residues dissolved in mobile phase before injection.
- Conditions: The dimensions of the columns were 100 x 2 mm with a flow rate of 0.2/0.3 mL/min. The dimensions of the column used for the quantitation were 125 x 3 mm with a flow rate of 0.4-0.5/0.5 mL/min.
- Detection: UV
- Recoveries: The overall recovery percentages were 80 and 117 %
- Results: The content of the test material in the experimental diet was found to be in good agreement with the nominal concentrations.
Duration of treatment / exposure:
Over two generations (until weaning of the F2 generation)
Frequency of treatment:
Daily (experimental diet was available ad libitum)
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 4 days of age.
Remarks:
Doses / Concentrations:
0, 30, 300, 1000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
30 males and 30 females per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: doses were selected on the basis of results from a 3-month oral toxicity study in which rats were fed 20, 100, 300, 1000 or 4000 ppm test material in the diet. During the study there were 3 deaths in the male group dosed at 4000 ppm. Body weight gains were reduced by 17 % and 41 % in males and 12 % and 20 % in females dosed at 1000 and 4000 ppm, respectively. Haematology and blood chemistry parameters were affected at 1000 and 4000 ppm. Urinary levels of bilirubin and urobilirubin were increased at doses of 300 ppm and above. Liver and spleen weights were increased at 4000 ppm. The liver was confirmed microscopically as the target organ. There were areas of diffuse necrosis, necrosis of single hepatocytes, inflammatory cell infiltration, pigmentation of hepatocytes and Kupffer cells, cytoplasmic vacuolisation, extramedullary haematopoiesis, and hepatocellular hypertrophy at 1000 and 4000 ppm. The maximum tolerated dose was met or exceeded at 1000 ppm and clearly exceeded at 4000 ppm. The doses selected for this study were therefore 0, 30, 300 and 1000 ppm.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily checks for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes (measured weekly)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):
Estrous cycle stages were recorded daily during the mating period until there was positive evidence of mating.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and subjected to gross necropsy.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities and developmental landmarks (righting reflex and eye opening)

GROSS EXAMINATION OF DEAD PUPS: Yes
The gross necropsy examination consisted of a macroscopic examination of the body, limbs, and organs of the thoracic and abdominal cavities, with special attention directed to the organs of the reproductive system.
Postmortem examinations (parental animals):
SACRIFICE
- All parent animals were necropsied after weaning and subjected to macroscopic examination, with histopathology of the sex and target organs

GROSS NECROPSY
- Gross necropsy consisted of examination of the following for gross pathological changes: vagina, uterus, ovaries, testes, epididymides, seminal vesicles, mammary area, prostate, pituitary gland, skin, spleen, mesenteric lymph node, axillary lymph node, popliteal lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary glands, liver, pancreas, oesophagus, stomach, small intestine, large intestine, kidneys, urinary bladder, adrenals, thyroid with parathyroid, thymus, peripheral nerve, brain, spinal cord, eyes with optic nerves, orbital glands, extraorbital lacrimal glands, zymbal glands, muzzle, tongue and all gross lesions

ORGAN WEIGHTS
- The following were weighed: ovaries, testes, spleen, heart, liver, kidneys, adrenals, thymus and brain

HISTOPATHOLOGY
- The following were prepared for microscopic examination for all animals of the control and high dose F0 and F1 animals selected for mating: vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate, pituitary gland, liver, mammary area and all gross lesions.
- In addition, the following organs were histopathologically examined in all groups: ovaries in non-pregnant positively mated females; testes, epididymides, seminal vesicles and prostate in males that failed to mate
- Additionally, to clarify the apparent differences in organ weights, the following organs were examined:
> F0 generation: liver (groups 2 and 3, all animals, both sexes), ovaries (groups 2 and 3, all females), spleen (groups 1 and 4, all males), heart (groups 1 and 4, all animals, both sexes), thymus (groups 1 and 4, all animals, both sexes), adrenals (groups 1 and 4, all males)
> F1 generation: liver (groups 2 and 3, all animals, both sexes), ovaries (groups 2 and 3, all females), adrenal (groups 1 and 4, all females)
Postmortem examinations (offspring):
SACRIFICE
- All pups in both generations (F1 pups not culled or selected for mating and F2 pups) were killed on or shortly after weaning of the last litter of that mating.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of a macroscopic examination of the body, limbs, and organs of the thoracic and abdominal cavities, with special attention directed to the organs of the reproductive systems (pups that were found dead or moribund were also subjected to the same procedure).
Statistics:
Statistical analysis of continuous data was performed using the Analysis of Variance Procedure (ANOVA) followed by Dunnett's t-test in case of a significant result. Categorical data were analysed using Chi-square test followed by Fischer's Exact test in case of a significant result. Non-parametric data were analysed using the Kruskal-Wallis nonparametric analysis of variance test followed by the Dunn test.
All microscopic findings from F0 and F1 animals were subjected to statistical analysis using statistical software. Over all findings Cochran Armatage’s linear trend test was performed. In all cases where an effect was found, further analyses were carried out by deleting the highest dose group and re-performing the analysis until a non-significant result was obtained.
Reproductive indices:
Female mating: number of females positively mated as a percentage of the number of females used for mating
Female fertility (=fecundity): number of females pregnant as a percentage of the females mated
Male mating: number of males with positive mating as a percentage of the number of males used for mating
Male fertility: number of males inducing pregnancy as a percentage of the number of males with positive mating
Parturition: number of females with births as a percentage of the number of females with confirmed pregnancy
Gestation: number of females with liveborn as a percentage of the number of females with confirmed pregnancy
Offspring viability indices:
Live birth: mean number of pups born alive per litter as a percentage of the mean number of pups born per litter
Viability: mean number of pups alive on day 4 (pre-culling) per litter as a percentage of the mean number of pups born alive per litter
Lactation: mean number of pups alive on day 21 per litter as a percentage of the mean number of pups alive on day 4 (post-culling) per litter
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
P Generation:
There were no treatment-related clinical signs and no treatment-related deaths among parental animals. One group 2 male (30 ppm) was found dead on day 111. Hypothermia, dyspnoea and hypoactivity had been noted for one day; haemorrhagic contents in the thoracic cavity was observed at necropsy. One group 4 female (1000 ppm) was found dead on gestation day 7. This female was pregnant, had no clinical signs and necropsy did not reveal any pathological findings. Incidental clinical signs observed occasionally among surviving animals included hair loss, wounds and/or crust, scurf, and chromodacryorrhea.
F1 Generation:
Furthermore, there were no treatment-related clinical signs among the F1 parent animals. Incidental clinical signs observed occasionally among surviving F1 animals included palpable masses, vaginal bloody discharge, hair loss and/or crust, and chromodacryorrhea.
There were no treatment-related deaths among the F1 parent animals. One 30 ppm male was found dead on day 43. At necropsy, dilatation and fibrinous adhesion of the small intestine were seen.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
P Generation:
During the premating period, males dosed at 300 and 1000 ppm, had 11 % and 22 % lower body weight gain compared to controls. Body weights were statistically significant from controls from day 68 and day 22, respectively. At 30 ppm for both sexes and at 300 and 1000 ppm for females, body weights and body weight gains were similar to control values.
For males at 1000 ppm, consumption was reduced by approximately 10 % from the start of the dosing period and differences from the control were statistically significant throughout the entire P generation. At 300 ppm, for males, differences from the control value attained statistical significance during weeks 2, 4 and 5 of the premating period; during the rest of the premating period, differences from the control value were minimal.
At 30 ppm, for both sexes and at 300 and 1000 ppm for females, food consumption was similar to that of the control group throughout the P generation. The only difference from the control value which attain statistical significance was for the females at 1000 ppm during the first week of the dosing period; differences from the control were minimal.
F1 Generation:
Group 300 and 1000 ppm F1 parent animals had significantly lower body weights on day 2, the first day of the F1 generation. Throughout the F1 generation, bodyweights in both groups remained lower than controls, but body weight gain was unusually similar to that of the control group.
Body weights for females were approximately 94 % of the controls (at 300 and 1000 ppm) during the premating period, weight gains were similar to controls.
In the low dose group (30 ppm), bodyweights and bodyweight gain were similar to that of the control group throughout the F1 generation. The only statistically significant changes were for the lower bodyweight and weight gain values for the males at 30 ppm on day 15 and during the second week of treatment (Days 8 -15) respectively.
Food consumption was reduced for both sexes at 300 and 1000 ppm throughout the premating period compared to the controls. This effect was more pronounced in males than in females. Food consumption for the females.
Food consumption in the females was similar in all groups throughout the gestation and lactation period.
Mean food consumption at 30 ppm for both sexes was similar to the control group throughout the F1 generation with the exception of a minimal reduction during the second week in males and an increase during weeks 6 and 10 in females.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
P Generation:
Mean test material intake for the P males reduced as bodyweight increased. At the start of the study, it was 0, 3.3, 321.9 and 101.6 mg/kg bw/day in the 0, 30, 300 and 1000 ppm dose groups, respectively. By the end of the treatment period, substance intake had reduced to approximately half these initial values. Substance intake for the P females was similar to that of the males at the start of the study - 0, 3.1, 30.0 and 91.9 mg/kg bw/day in the 0, 30, 300 and 1000 ppm dose groups, respectively. By the end of the premating period, substance intake had reduced by up to 30 %. During gestation, substances intake was stable at values similar to those found at the end of the premating period. In contrast, during lactation, there was a marked increase in substance intake as a result of increased food intake by the dams and the pups feeding. At the end of the lactation period, values were 0, 6.3, 64.0 and 215.8 mg/kg bw/day in the 0, 30, 300 and 1000 ppm dose groups, respectively.
F1 Generation:
As in the P generation, mean test material intake reduced as body weight increased. In the first week of the F1 generation, values were 0, 4.1, 42.5 and 146.9 mg/kg bw/day for males and 0, 3.8, 39.7 and 143.1 mg/kg bw/day for females in 0, 30, 300 and 1000 ppm groups, respectively. By the end of the pre-mating period, substance intake had reduced by between 55 % and 65 %. For the females, substance intake remained stable during the gestation period but there was a marked increase during the lactation period as a result of increased food intake by the dams and the pups feeding. At the end of the lactation period, values were 0, 6.2, 65.4 and 209.4 mg/kg bw/day in the 0, 30, 300 and 1000 ppm groups, respectively. In comparison with the P generation, values for test material intake at the start of the F1 generation were higher because the animals were slightly younger and weighed less.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
P Generation:
There was no treatment-related effects on the number of animals mating, the number of females becoming pregnant or on the mean pre-coital time. Only 7 males failed to mate (one control, two 30 ppm, one 300 ppm and three 1000 ppm). Three mated females were not pregnant (one control, one 300 ppm and one 1000 ppm).
The mean duration of gestation was approximately 22 days in all groups. There was no indication of an adverse effect of treatment on any of the gestation or parturition indices. A total of 27, 28, 27 and 25 females in groups 1 to 4 were pregnant and gave birth to live young.
F1 Generation:
There were no treatment-related effects on the number of animals mating, the number of females becoming pregnant or on the mean pre-coital time.
Mating generally occurred within one estrous cycle for all mated females. Four, seven, two and three mated females were not pregnant in the control to 1000 ppm group.
There was no indication of an adverse effect of treatment on any of the gestation or parturition indices. A total of 26, 27 and 25 females in groups 1 to 4, respectively were pregnant and gave birth to live young. A total of four mated females (one group 2 female, one group 3 female and two group 4 females) did not deliver, but were pregnant by Salawski stain at necropsy.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
For all mated P females, mating generally occurred within one estrous cycle.

ORGAN WEIGHTS (PARENTAL ANIMALS)
P Generation:
At 300 amid 1000 ppm, exsanguinated body weights for males were lower (6 % and 13 %, respectively) and significantly different from controls. At 1000 ppm, most of the absolute organ weights were also lower, and the differences were significant for spleen, liver, adrenals, and thymus. At 300 ppm, absolute spleen and liver weights were significantly lower than controls. In contrast, at both dose levels, the majority of relative organ weights were similar or higher than those of the control group. At 1000 ppm heart, kidney, testes and brain were significantly higher than controls. At 300 ppm, heart and brain relative weights were significantly higher than controls and liver relative weights were slightly lower than controls. These findings were considered to be due to the treatment-related effect of body weight and not to be a specific toxic effect on target organs.
F1 generation:
Exsanguinated bodyweights at 300 and 1000 ppm males were approximately 64 and 12 % lower respectively and significantly different from controls. At 1000 ppm most of the absolute organ weights were lower and the differences were significant for heart, liver and kidneys. At 300 ppm, some absolute organ weights were lower, but none of the differences were statistically significant.
In contrast, most of the relative organ weights were higher that the control. Spleen, heart, testes, thymus and brain were significantly higher at 1000 ppm and testes and brain at 300 ppm. These findings were considered to be a direct result of the reduced bodyweight and not specifically a toxic effect on target organs. All other changes were found to be within the historical control range and were not accompanied by any histopathological changes.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related macroscopic changes were observed at terminal necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS)
P Generation:
No treatment-related microscopic changes were observed following examination of the reproductive organs of the control and high dose groups or at microscopic examination of heart, adrenal, ovaries, spleen and thymus from selected groups. Treatment-related statistically significant findings were confined to the liver of 300 and 1000 ppm groups, and included: minimal/mild bile duct hyperplasia in males and females at 300 and 1000 ppm; minimal hepatocelular hypertrophy in males at 1000 ppm; and minimal/mild foci of necrosis in occasional animals (males at 300 and 1000 ppm, and females at 1000 ppm).
F1 Generation:
No treatment-related microscopic changes were observed at microscopic examination of the reproductive organs and adrenals (females) of the control and high dose groups or at microscopic examination of ovaries from selected groups. Treatment-related statistically significant findings were confined to the liver of 300 and/or 1000 ppm groups, and included: minimal/mild bile duct hyperplasia in males and females at 300 and 1000 ppm; minimal hepatocellular hypertrophy in males at 1000 ppm; and minimal/mild foci of necrosis in occasional animals (males at 300 and 1000 ppm and females at 1000 ppm).
Dose descriptor:
NOEL
Remarks:
(reprotoxic effects)
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment-related effects on any of the reproductive parameters
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOEL
Remarks:
(systemic effects)
Effect level:
30 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on body weight gain reduction and liver histopathology.
Remarks on result:
other: Generation: P and F1 (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
LITTER INDICES
F1 Pups:
At birth, mean litter size was comparable in all groups. At 300 ppm, the incidence of pup loss between days 1 and 4 post-partum, prior to culling, was higher than that of the control group. After culling on day 4, mean litter size and pup loss during the lactation period were similar in all groups to that of the control group. The sex ratios of the F1 pups on days 0 to 21 post-partum were similar in all groups.
F2 Pups:
All F1 pregnant females which gave birth reared their young to weaning on day 21 post partum. In all test groups, both the viability index and the lactation index, mean litter size and pup loss during the lactation period were similar to that of the control group. The sex ratios of the pups on days 0 and 21 post partum were similar in all groups.

VIABILITY (OFFSPRING)
F1 Pups:
Differences from the control value attained statistical significance for the lower percentage of pups surviving days 0 to 4 however this was attributed to a dam failing to wean her litter (not treatment related)
F2 Pups:
The percentage of pups surviving days 0 to 4 were similar to the control group.

CLINICAL SIGNS (OFFSPRING)
F1 Pups:
During lactation, there were no clinical signs for the F1 pups which were considered to be related to treatment.
F2 Pups:
During lactation, there were no clinical signs for the F2 pups which were considered to be related to treatment of the dams with the test material.

BODY WEIGHT (OFFSPRING)
F1 Pups:
At 300 and 1000 ppm, from birth to day 14, mean F1 pup weight was similar to that of the control group. However, there was a retardation in weight gain in both groups, particularly between days 14 and 21, such that mean pup bodyweights were lower than controls on day 21 by up to 9 % in the 1000 ppm group.
F2 Pups:
Mean body weight of F2 pups was similar in all groups. At 1000 ppm there was an approximate 10 % retardation in weight gain between days 7 and 21 post partum.

GROSS PATHOLOGY (OFFSPRING)
No treatment-related findings were noted at necropsy of the F1 of F2 pups.
Reproductive effects observed:
not specified

Table 1: Mean body weights for P Males

Dose (ppm)

0

30

300

1000

Day

1

219.9

218.8

217.8

218.5

8

278.3

273.3

271.9

269.6

15

328.3

321.3

317.0

314.3

22

368.4

358.6

352.0

346.4*

29

399.5

388.4

379.3

370.7**

36

426.3

412.9

403.2

391.9**

43

447.8

434.4

424.1

411.1**

51

466.1

453.4

441.4

424.0**

57

479.8

466.1

453.2

432.9**

64

491.4

478.8

464.5

439.7**

68

499.7

486.3

470.0*

444.4**

71

493.7

483.3

469.6

439.3**

78

503.1

491.3

471.9*

451.0**

85

517.2

504.1

485.3*

458.7**

92

533.0

519.9

498.1*

467.7**

99

542.3

532.4

508.6*

474.3**

106

555.2

540.4

518.7*

483.9**

113

566.1

550.5

526.9*

487.7**

120

573.0

554.6

532.2*

491.8**

127

577.8

560.6

537.1*

497.5**

134

589.5

571.0

545.8*

504.6**

* = p < 0.05

** = p < 0.01

Table 2: Mean body weights P females

Dose (ppm)

0

30

300

1000

Day

Premating

1

162.1

163.2

160.8

163.9

8

188.7

192.1

188.8

188.3

15

211.4

213.4

210.8

207.9

22

227.9

230.5

227.0

227.8

29

240.7

245.9

241.8

240.5

36

252.3

254.2

249.2

247.9

43

260.9

268.5

260.1

256.5

51

268.2

274.3

267.0

262.7

57

275.5

280.2

272.9

266.0

64

279.2

285.0

276.9

271.0

68

280.6

286.9

279.4

274.6

71

280.4

289.1

281.9

275.3

Gestation

0

278.8

286.7

281.1

274.8

7

300.9

307.6

301.5

296.5

14

331.4

340.0

332.6

327.2

21

423.0

437.4

428.7

415.9

Lactation

0

316.5

330.1

318.6

309.0

7

326.6

336.5

329.2

323.7

14

339.7

349.9

342.0

336.5

21

333.6

342.4

337.1

335.4

Table 3: Mean body weights F1 males

Dose (ppm)

0

30

300

1000

Day

2

185.6

176.7

166.0**

153.3**

8

234.6

224.4

211.6**

197.2**

15

293.2

279.8*

264.8**

247.3**

22

341.5

328.9

311.4**

294.4**

29

376.5

367.4

348.6**

332.0**

36

405.2

396.1

376.7**

355.3**

43

433.7

423.7

404.3**

381.4**

50

452.9

444.7

424.0**

398.3**

57

467.9

463.0

438.4**

410.0**

64

479.4

475.6

450.9*

420.0**

68

488.63

485.2

453.2**

424.1**

71

486.3

481.7

454.3**

411.8**

78

489.6

483.7

457.9**

425.4**

85

503.9

498.8

470.6**

433.7**

92

520.8

513.2

483.8**

449.8**

99

529.6

523.7

495.7*

461.3**

106

543.1

539.6

507.8*

474.4**

113

533.7

550.2

518.2*

482.5**

120

559.9

556.3

523.3*

487.6**

* = p < 0.05

** = p < 0.01

Table 4: Mean body weights F1 females

Dose (ppm)

0

30

300

1000

Day

Premating

2

151.4

144.6

133.4**

130.7**

8

174.9

168.3

160.3**

156.7**

15

200.5

195.5

185.8**

182.5**

22

221.8

219.4

206.9*

204.4**

29

238.9

234.6

224.9*

221.8**

36

249.5

249.7

237.1

236.5

43

263.6

263.4

250.0

247.4*

50

273.4

273.8

261.0

258.0

57

282.2

279.8

266.9

264.3*

64

286.3

185.3

274.2

271.9

68

290.2

288.7

277.0

273.5

71

292.9

292.4

276.0

275.7

Gestation

0

281.1

284.4

275.5

270.7

7

305.1

307.2

297.1

292.1

14

336.0

339.2

326.9

322.3

21

427.4

432.4

412.1

411.8

Lactation

0

316.7

323.4

308.3

302.2

7

330.3

334.3

323.4

323.6

14

345.3

347.1

335.9

338.4

21

342.3

338.6

330.1

333.8

* = p < 0.05

** = p < 0.01

Table 5: Incidence of treatment-related liver findings

P Generation

Dose (ppm)

Males

Females

0

30

300

1000

0

30

300

1000

Hepatocelular hypertrophy (minimal)

0

0

0

0

0

0

0

0

Bile duct hyperplasia (minimal/mild)

2

1

10*

19*

0

1

4*

8*

Foci of necrosis (minimal/mild)

0

0

1

3*

0

0

0

1

F1 Generation

Dose (ppm)

Males

Females

0

30

300

1000

0

30

300

1000

Hepatocelular hypertrophy (minimal)

0

0

0

11*

0

0

0

0

Bile duct hyperplasia (minimal/mild)

1

4

11*

19*

1

1

6*

8*

Foci of necrosis (minimal/mild)

0

0

2*

5*

0

0

0

2*

* = p < 0.05

Conclusions:
Under the conditions of the study, none of the reproductive parameters - gonadal function, mating behaviour, conception, parturition, lactation and weaning - were affected by the administration of the test material to the parent animals at any dose level, in either the P or F1 generation. Based on the results of this study there was no evidence of reproductive toxicity through 1000 ppm. Based on the body weight gain reduction and liver histopathology, the no observed effect level for systemic toxicity was 30 ppm. The no observed effect level for reproductive toxicity was determined to be 1000 ppm.
Executive summary:

The reprotoxicity of the test material was investigated in accordance with standardised guidelines OECD 416, EC OJ No L133/47, Japanese MAFF 59 No 4200 and EPA OPP 83 -4. In two successive generations (P and F1), male and female rats were continuously exposed to the test material admixed in diet at nominal concentrations of 0, 30, 300 and 1000 ppm. At 10 weeks premating dietary exposure to the test material, animals were paired 1:1 within each dose group (30 animals per sex and dose) until there was evidence of positive mating or for 19 days, whichever occurred first. Dams were allowed to litter and suckle their pups naturally. Litter were culled to 4 male and 4 female pups, where possible, on day 4 post partum. After weaning, selected F1 young (30 animals per sex and dose) were continuously exposed to the test material in the diet as in the P generation. After 10 weeks exposure, these F1 animals w4ere cohabited 1:1 within each dose group for up to 19 days. Resulting F2 litters were necropsied after weaning. Clinical signs, body weights, food consumption, mating, gestation and delivery parameters, pup survival and physical and behavioural developmental landmarks were recorded. A gross necropsy examination was performed on all pups not selected for mating. All parent animals were necropsied after weaning and subjected to macroscopic examination, with histopathology of the sex and target organs.

There were no treatment-related clinical signs or treatment-related deaths among the P animals. At 300 and 1000 ppm, for P males, food consumption was reduced and body weight gain was retarded from the start of the dosing period. As a result, body weights were significantly lower than those of the control group. Male and female mating and fertility indices, maternal gestation and parturition indices and the duration of gestation were unaffected by treatment. Mean weight gain of the pups was retarded during lactation days 14 to 21 at 300 ppm (93 % of control) and 1000 ppm (84 % of control); and body weights were reduced at weaning. For all test groups, no treatment-related effects were noted in the F1 offspring in terms of the sex ratios, clinical signs, or macroscopic findings at necropsy. For P parent animals there were no treatment-related findings at terminal necropsy, in organ weights or in the histopathological examination of the adrenals, thymus, spleen, heart or reproductive organs. Microscopic examination of the liver revealed increased incidence of males and females at 300 and 1000 ppm with bile duct hyperplasia, hepatocellular hypertrophy and necrosis.

There were no treatment-related clinical signs or treatment-related deaths among the F1 parent animals. At 300 and 1000 ppm, body weights of the selected F1 animals were significantly lower than those of the control group. During the premating period, for both sexes, body weights and food consumption in these groups were significantly lower than controls, but body weight gain was unusually similar to that of the control group. Male and female mating and fertility indices, maternal gestation and parturition indices and the duration of gestation were unaffected by treatment. At 1000 ppm, mean pup body weight gain was retarded by approximately 10 % during lactation days 7 to 21 and body weights were reduced at weaning. At 30 and 300 ppm, pup weights from birth, through lactation to weaning were no affected by treatment. For all groups, litter size, post-natal loss were not affected by treatment. For all test groups, no treatment-related effects were noted in the F2 offspring in terms of the sex ratios, the development of physical landmarks, clinical signs, or the macroscopic findings noted at necropsy. For F1 parent animals, there were no treatment-related findings at terminal necropsy, in the organ weights or in the histopathological examination of the adrenals or reproductive organs. Microscopic examination of the liver showed similar findings as in P parents.

At 300 and 1000 ppm, food consumption was reduced and body weight gain was retarded in both P and F1 males and in F1 females. Additionally, at both of these dose levels, in both generations, body weight gain of the pups was retarded during the lactation period. Increased incidence of liver pathology were observed in males and females in the P and F1 generations - minimal/mild bile duct hyperplasia in both sexes at 300 and 1000 ppm, minimal hepatocellular hypertrophy in males at 1000 ppm, and minimal/mild foci of necrosis at 300 and 1000 ppm and in few females at 1000 ppm.

None of the reproductive parameters - gonadal function, mating behaviour, conception, parturition, lactation and weaning - were affected by the administration of the test material to the parent animals at any dose level, in either the P or F1 generation. Based on the results of this study there was no evidence of reproductive toxicity through 1000 ppm. Based on the body weight gain reduction and liver histopathology, the no observed effect level for systemic toxicity was 30 ppm. The no observed effect level for reproductive toxicity was determined to be 1000 ppm.

The available data are deemed to be relevant, reliable and adequate for the purposes of risk assessment.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
rat
Quality of whole database:
The study was conducted to GLP and in accordance with standardised guidelines with a high level of reporting. The study was assigned a reliability score of 1 according to the criteria of Klimisch (1997) and considered suitable as an accurate reflection of the test material.
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 reprotoxicity of the test material was investigated in accordance with standardised guidelines OECD 416, EC OJ No L133/47, Japanese MAFF 59 No 4200 and EPA OPP 83 -4. In two successive generations (P and F1), male and female rats were continuously exposed to the test material admixed in diet at nominal concentrations of 0, 30, 300 and 1000 ppm. At 10 weeks premating dietary exposure to the test material, animals were paired 1:1 within each dose group (30 animals per sex and dose) until there was evidence of positive mating or for 19 days, whichever occurred first. Dams were allowed to litter and suckle their pups naturally. Litter were culled to 4 male and 4 female pups, where possible, on day 4 post partum. After weaning, selected F1 young (30 animals per sex and dose) were continuously exposed to the test material in the diet as in the P generation. After 10 weeks exposure, these F1 animals w4ere cohabited 1:1 within each dose group for up to 19 days. Resulting F2 litters were necropsied after weaning. Clinical signs, body weights, food consumption, mating, gestation and delivery parameters, pup survival and physical and behavioural developmental landmarks were recorded. A gross necropsy examination was performed on all pups not selected for mating. All parent animals were necropsied after weaning and subjected to macroscopic examination, with histopathology of the sex and target organs.

There were no treatment-related clinical signs or treatment-related deaths among the P animals. At 300 and 1000 ppm, for P males, food consumption was reduced and body weight gain was retarded from the start of the dosing period. As a result, bodyweights were significantly lower than those of the control group. Male and female mating and fertility indices, maternal gestation and parturition indices and the duration of gestation were unaffected by treatment. Mean weight gain of the pups was retarded during lactation days 14 to 21 at 300 ppm (93 % of control) and 1000 ppm (84 % of control); and body weights were reduced at weaning. For all test groups, no treatment-related effects were noted in the F1 offspring in terms of the sex ratios, clinical signs, or macroscopic findings at necropsy. For P parent animals there were no treatment-related findings at terminal necropsy, in organ weights or in the histopathological examination of the adrenals, thymus, spleen, heart or reproductive organs. Microscopic examination of the liver revealed increased incidence of males and females at 300 and 1000 ppm with bile duct hyperplasia, hepatocellular hypertrophy and necrosis.

There were no treatment-related clinical signs or treatment-related deaths among the F1 parent animals. At 300 and 1000 ppm, body weights of the selected F1 animals were significantly lower than those of the control group. During the premating period, for both sexes, body weights and food consumption in these groups were significantly lower than controls, but body weight gain was unusually similar to that of the control group. Male and female mating and fertility indices, maternal gestation and parturition indices and the duration of gestation were unaffected by treatment. At 1000 ppm, mean pup bodyweight gain was retarded by approximately 10 % during lactation days 7 to 21 and body weights were reduced at weaning. At 30 and 300 ppm, pup weights from birth, through lactation to weaning were no affected by treatment. For all groups, litter size, post-natal loss were not affected by treatment. For all test groups, no treatment-related effects were noted in the F2 offspring in terms of the sex ratios, the development of physical landmarks, clinical signs, or the macroscopic findings noted at necropsy. For F1 parent animals, there were no treatment-related findings at terminal necropsy, in the organ weights or in the histopathological examination of the adrenals or reproductive organs. Microscopic examination of the liver showed similar findings as in P parents.

At 300 and 1000 ppm, food consumption was reduced and body weight gain was retarded in both P and F1 males and in F1 females. Additionally, at both of these dose levels, in both generations, body weight gain of the pups was retarded during the lactation period. Increased incidence of liver pathology were observed in males and females in the P and F1 generations - minimal/mild bile duct hyperplasia in both sexes at 300 and 1000 ppm, minimal hepatocellular hypertrophy in males at 1000 ppm, and minimal/mild foci of necrosis at 300 and 1000 ppm and in few females at 1000 ppm.

None of the reproductive parameters - gonadal function, mating behaviour, conception, parturition, lactation and weaning - were affected by the administration of the test material to the parent animals at any dose level, in either the P or F1 generation. Based on the results of this study there was no evidence of reproductive toxicity through 1000 ppm. Based on the body weight gain reduction and liver histopathology, the no observed effect level for systemic toxicity was 30 ppm. The no observed effect level for reprotoxicity was determined to be 1000 ppm.


Short description of key information:
NOEL (reprotoxic effects) = 1000 ppm; NOEL (systemic effects) = 30 ppm, rat (male/female), OECD 416, EC OJ No L133/47, Japanese MAFF 59 No 4200 and EPA OPP 83 -4, Khalil 1998

Justification for selection of Effect on fertility via oral route:
Only one study is available

Effects on developmental toxicity

Description of key information
NOEL 1000 mg/kg bw/day (maternal and developmental), rat oral teratogenicity, OECD 414, EC OJ No L133/24, Japanese MAFF 59 and US EPA OPP 83 -3, Khalil 1996a
NOEL 100 mg/kg bw/day (maternal and developmental), rabbit oral teratogenicity, OECD 414, EC OJ No L133/24, Japanese MAFF 59 and US EPA OPP 83 -3, Khalil 1996b
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August 1995 to 19 February 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed to GLP and in line with the standardised guidelines OECD 414, EC OJ No L133/24, Japanese MAFF 59 and US EPA OPP 83 -3, with no deviations thought to impact the reliability of the presented results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese MAFF 59 NohSan No. 4200, "Teratogenicity Study" January 1985
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: European Communities Commission Directive 87/302/EEC, "Teratogenicity Study - Rodent and Non-Rodent", OJ No L133/24, May 30, 1988
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Species:
rabbit
Strain:
other: Russian Chbb:HM
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 3 months (minimum)
- Fasting period before study: Not reported
- Housing: housed individually in battery cages with steel slatted floors.
- Diet (e.g. ad libitum): Pelleted, certified standard feed, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 2 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): about 16 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 11/09/95 To: 29/02/96
Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxymethylcellulose and 0.1 % aqueous polysorbate 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance-vehicle mixtures were prepared daily with a high-speed homogeniser. Homogeneity of the mixtures during administration was maintained with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 2.5, 25 and 250 mg/mL mixture
- Amount of vehicle (if gavage): 4 mL/kg of actual bodyweight
- Purity: 0.5% (w/w) aqueous solution of sodium carboxymethylcellulose and 0.1% (w/w) aqueous polysorbate 80
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In order to permit determination of content, homogeneity and stability of the test substance under the actual conditions of administration during the study, samples of test substance-vehicle mixtures were taken on the date(s) designated below, once before and once after dosing. The samples from before dosing were taken from the top, middle and bottom of the container; the samples from after dosing were taken from the middle of the container.
Samples were taken in duplicate. Together with 10 mL of vehicle and approximately 2.0 g of test substance they were frozen until analysis.
- dates of sampling: 21 and 27 September 1995

Samples were analysed by HPLC with the following conditions:
- Column: 5 µm; 125 mm x 4.6 mm (o.d.)
- Temperature: room temperature
- Eluent: Acetonitrile/0.02 M H3PO4 (60+40 v/v)
- Flow: 1.0 mL/min
- Wavelength: 274 nm
- Injection volume: 10 μL
Details on mating procedure:
- Impregnation procedure: Females were artificially inseminated with diluted semen from bucks of the same strain. The day of insemination was designated as day 0 of pregnancy.
Duration of treatment / exposure:
From day 7 to day 19 of gestation.
Frequency of treatment:
Daily
Duration of test:
29 days
Remarks:
Doses / Concentrations:
0, 10, 100 and 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
20 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a previous rangefinding study in pregnant rabbits (study no. 951036). In the preliminary study, test substance was non-toxic to dams, embryos and foetuses at doses up to 1000 mg/kg, and there was no indication of teratogenesis.
- Rationale for animal assignment (if not random): Inseminated females were allocated to experimental and control groups using a method of randomisation by weight stratification.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (> 6 hours apart)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes (on days 4, 7, 12, 16, 20, 24 and 29)
- Food consumption for each animal determined according to the following formula: feed comsumption (g) per period / days per period

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Macroscopic pathological examination of the main organs of the thoracic and abdominal cavities, in particular the genitals.
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 of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all of litter
- Visceral examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Statistical analysis of continuous data (e.g. body weight, feed consumption) was performed using the Analysis of Variance Procedure (ANOVA) followed by Dunnett's t-Test in case of a significant result in the ANOVA. Categorical data (e.g. malformation counts) were analysed using Chi-Square test followed by Fisher's Exact test in case of a significant result in the Chi-Square test.
Non-parametric data (e.g. mean percent affected fetuses/litter) were analysed using the Kruskal-Wallis nonparametric analysis of variance test followed by Mann-Whitney U-test.
Historical control data:
Available and documented within the study report.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- Maternal Toxicity
None of the maternal animals died during the study and only occasional incidental clinical signs occurred during the study.
At 1000 mg/kg, there was a slight reduction in food consumption during the dosing period (days 7 to 20) and an increase in food consumption at the end of gestation when the animals were no longer dosed (days 20 to 29). In this group a significant difference in weight change was seen only for days 12 to 16 of gestation and overall on days 7 to 19. Overall weight change for days 7 to 29 was comparable among all groups.
Gravid uterus weights, carcass weights, and net weight change from day 7 were similar in all four groups. There were no maternal necropsy findings.

- Reproduction and Cesarean Section Data
Preimplantation losses, numbers of implantation sites and live litter size were similar in all groups.
At 1000 mg/kg, there was an increase in the incidence of post implantation loss, almost exclusively in the form of early resorptions. Three females in this group showed total early embryonic resorption; for the remaining 16 pregnant females, this parameter was increased when compared to controls and historical control data.
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
At 1000 mg/kg, foetal weights (both male and female) were slightly reduced compared to controls.
The incidence and type of external, visceral and skeletal findings were not affected by treatment.
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Analytical results:

The chemical analysis determined the concentration, homogeneity and stability of the test material in 0.1% (w/w) aqueous polysorbate 80 with 0.5% (w/w) CMC by HPLC-analysis. The overall mean concentration of the homogeneity samples were found to be 92.7%, 92.6%, and 99.0% of the nominal concentrations for dose group 2 (2.5 mg/mL), for dose group 3 (25 mg/mL), and for dose group 4 (250 mg/mL), respectively. The homogeneity varied in the range from -2% to +2% of the mean concentrations. The test material was found to be stable in 0.1% (w/w) aqueous polysorbate 80 with 0.5% (w/w) CMC under actual conditions of administration over the period of dosing.

Table 1: Mean Maternal Bodyweight (g)

Day

Mean Maternal Bodyweight (g)

Group 1

(0 mg/kg bw/day)

Group 2

(10 mg/kg bw/day)

Group 3

(100 mg/kg bw/day)

Group 4

(1000 mg/kg bw/day)

0

2882

2874

2878

2892

1

2896

2890

2875

2903

2

2888

2886

2878

2902

3

2876

2887

2870

2887

4

2878

2869

2864

2882

5

2865

2865

2860

2870

6

2858

2857

2854

2865

7

2860

2855

2854

2860

8

2851

2856

2845

2863

9

2843

2851

2850

2849

10

2851

2854

2842

2842

11

2846

2844

2841

2840

12

2837

2853

2844

2828

13

2836

2864

2847

2819

14

2842

2866

2849

2829

15

2856

2873

2861

2821

16

2860

2884

2870

2818

17

2866

2888

2868

2803

18

2857

2887

2869

2790

19

2853

2879

2863

2782

20

2842

2869

2847

2763

21

2836

2866

2848

2755

22

2833

2861

2840

2763

23

2839

2864

2843

2781

24

2851

2879

2851

2805

25

2870

2882

2868

2828

26

2884

2898

2876

2843

27

2902

2906

2889

2871

28

2917

2924

2901

2902

29

2932

2943

2907

2921

 

Table 2: Mean Maternal Bodyweight Gain (g)

Days

Mean Maternal Bodyweight Change (g)

Group 1

(0 mg/kg bw/day)

Group 2

(10 mg/kg bw/day)

Group 3

(100 mg/kg bw/day)

Group 4

(1000 mg/kg bw/day)

0-4

-4

-5

-14

-10

4-7

-19

-13

-10

-23

7-12

-23

-2

-9

-31

12-16

24

31

26

-11*

16-20

-18

-14

-23

-54

20-24

9

9

4

42

24-29

81

64

56

116

7-19

-7

23

9

-78*

7-29

72

88

53

61

 * p ≤ 0.05

Table 3: Mean Food Consumption

Days

Mean Food Consumption g/animal/day

Group 1

(0 mg/kg bw/day)

Group 2

(10 mg/kg bw/day)

Group 3

(100 mg/kg bw/day)

Group 4

(1000 mg/kg bw/day)

0-4

91.9

95.4

87.4

91.8

4-7

82.2

89.3

87.2

87.5

7-12

83.9

86.4

86.9

71.7

12-16

64.6

73.2

69.6

49.2

16-20

63.9

71.8

70.3

42.1

20-24

69.6

76.5

71.2

81.5

24-29

80.1

75.0

79.7

105.6*

 * p≤ 0.05

Table 4: Summary of Caesarean Section Data for Pregnant Dams

Observation

Group 1

(0 mg/kg bw/day)

Group 2

(10 mg/kg bw/day)

Group 3

(100 mg/kg bw/day)

Group 4

(1000 mg/kg bw/day)

Number of pregnant animals used for calculation

19

17

18

19

Corpora lutea

Total

121

120

122

132

Mean

6.4

7.1

6.8

6.9

Implantation Sites

Total

97

93

89

112

Mean

5.1

5.5

4.9

5.9

Preimplantation loss % per animal

Total

24

27

33

20

Mean %

21.3

23.1

27.5

17.2

Foetuses (per animal)

Total

83

71

77

81

Mean

4.4

4.2

4.3

4.3

% Alive

100

100

100

100

% Dead

0

0

0

0

Live foetuses (per animal)

Total

83

71

77

81

Mean

4.4

4.2

4.3

4.3

Malformed live foetuses (per animal)

Total

0

0

0

1

Mean

0

0

0

0.1

Dead foetuses

Total

0

0

0

0

Early resorptions (per animal)

Total

14

21

12

29

Mean

0.7

1.2

0.7

1.5

% of implantations per animal

16.5

21.3

12.7

31.9

Late resorptions (per animal)

Total

0

1

0

2

Mean

0

0.1

0

0.1

% of implantations per animal

0

1.5

0

1.4

Abortion Sites

Total

0

0

0

0

Postimplantation loss (per animal)

Total

14

22

12

31

Mean

0.7

1.3

0.7

1.6

% of implantations per animal

16.5

22.8

12.7

33.3

Affected implants (per animal)

Total

14

22

12

32

Mean

0.7

1.3

0.7

1.7

% of implantations per animal

16.5

22.8

12.7

34.0

Number of litters used for calculations

18

15

18

16

Viable male foetuses

Number

39

34

40

36

%

47.0

47.9

51.9

44.4

Viable female foetuses

Number

44

37

37

45

%

53.0

52.1

48.1

55.6

Foetal bodyweight (g)

Mean

41.4

41.0

41.9

40.0

Male foetal bodyweight (g)

Mean

41.3

41.5

42.4

39.8

Female foetal bodyweight (g)

Mean

41.5

39.6

41.4

40.2

 

Table 5: Summary of Caesarean Section Data Day 29 Post-Coitum

Observation

Group 1

(0 mg/kg bw/day)

Group 2

(10 mg/kg bw/day)

Group 3

(100 mg/kg bw/day)

Group 4

(1000 mg/kg bw/day)

Number of pregnant animals used for calculation

18

15

18

16

Corpora lutea

Total

116

106

122

119

Mean

6.4

7.1

6.8

7.4

Implantation Sites

Total

93

80

89

103

Mean

5.2

5.3

4.9

6.4

Preimplantation loss % per animal

Total

23

26

33

16

Mean %

21.4

25.2

27.5

13.7

Foetuses (per animal)

Total

83

71

77

81

Mean

4.6

4.7

4.3

5.1

% Alive

100

100

100

100

% Dead

0

0

0

0

Live foetuses (per animal)

Total

83

71

77

81

Mean

4.6

4.7

4.3

5.1

Malformed live foetuses (per animal)

Total

0

0

0

1

Mean

0

0

0

0.1

Dead foetuses

Total

0

0

0

0

Early resorptions (per animal)

Total

10

8

12

20

Mean

0.6

0.5

0.7

1.3

% of implantations per animal

11.9

10.8

12.7

19.2

Late resorptions (per animal)

Total

0

1

0

2

Mean

0.0

0.1

0.0

0.1

% of implantations per animal

0.0

1.7

0.0

1.7

Abortion Sites

Total

0

0

0

0

Postimplantation loss (per animal)

Mean

10

9

12

22

Total

0.6

0.6

0.7

1.4

% of implantations per animal

11.9

12.5

12.7

20.8

Affected implants (per animal)

Total

10

9

12

23

Mean

0.6

0.6

0.7

1.4

% of implantations per animal

11.9

12.5

12.7

21.6

Number of litters used for calculations

18

15

18

16

Viable male foetuses

Number

39

34

40

36

%

47.0

47.9

51.9

44.4

Viable female foetuses

Number

44

37

37

45

%

53.0

52.1

48.1

55.6

Foetal bodyweight (g)

Mean

41.4

41.0

41.9

40.0

Male foetal bodyweight (g)

Mean

41.3

41.5

42.4

39.8

Female foetal bodyweight (g)

Mean

41.5

39.6

41.4

40.2

Conclusions:
Under the conditions of the study, maternal toxicity (reduced food consumption and body weight gain) and embryo and fetal toxicity (increased incidence of early resorption and decreased fetal weights) were seen in the 1000 mg/kg group. The No Observed Effect Level (NOEL) of the test material in this study was therefore considered to be 100 mg/kg for dams and fetuses. There was no indication of teratogenic potential.
Executive summary:

The study was performed to meet the requirements of OECD 414, EC OJ No L133/24, Japanese MAFF 59 and US EPA OPP 83 -3 under GLP conditions. The study was performed to evaluate the potential of the test substance to cause embryotoxic, foetotoxic, and teratogenic effects in rabbits. The test substance was evaluated over a range of concentrations - 0, 10, 100 and 1000 mg/kg. Females were inseminated with semen from males of the same stock. Inseminated females were treated daily with the test material from day 7 to day 19 of gestation. On day 29 dams were sacrificed and foetuses extracted and examined for skeletal and visceral effects.

None of the maternal animals died during the study and only occasional incidental clinical signs occurred during the study.

At 1000 mg/kg, there was a slight reduction in food consumption during the dosing period (days 7 to 20) and an increase in food consumption at the end of gestation when the animals were no longer dosed (days 20 to 29). In this group a significant difference in weight change was seen only for days 12 to 16 of gestation and overall on days 7 to 19. Overall weight change for days 7 to 29 was comparable among all groups.

Gravid uterus weights, carcass weights, and net weight change from day 7 were similar in all four groups. There were no maternal necropsy findings.

Preimplantation losses, numbers of implantation sites and live litter size were similar in all groups.

At 1000 mg/kg, there was an increase in the incidence of post implantation loss, almost exclusively in the form of early resorptions. Three females in this group showed total early embryonic resorption; for the remaining 16 pregnant females, this parameter was increased when compared to controls and historical control data.

At 1000 mg/kg, foetal weights (both male and female) were slightly reduced compared to controls. However, the incidence and type of external, visceral and skeletal findings were not affected by treatment.

Under the conditions of the study, maternal toxicity (reduced food consumption and body weight gain) and embryo and foetal toxicity (increased incidence of early resorption and decreased foetal weights) were seen in the 1000 mg/kg group. The No Observed Effect Level (NOEL) of the test material in this study was therefore considered to be 100 mg/kg for dams and foetuses. There was no indication of teratogenic potential.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rabbit
Quality of whole database:
All four available studies were conducted in line with GLP and in accordance with standardised guidelines; all three studies were therefore assigned a reliability score of 1 according to the criteria of Klimisch (1997) and were considered suitable as an accurate reflection of the test material.
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

Khalil (1996a) evaluated the potential of the test material to cause embryotoxic, foetotoxic, and teratogenic effects in rats. The study was conducted to GLP and in accordance with standardised guidelines OECD 414, EC OJ No L133/24, Japanese MAFF 59 and US EPA OPP 83 -3. During the study, test material was administered to pregnant dams, daily, from day 6 to day 15 of gestation at 0, 10, 100 and 1000 mg/kg. On day 21 dams were sacrificed and foetuses extracted and examined for skeletal and visceral effects. There were no maternal clinical signs which were considered to be treatment-related and there were no premature mortalities. Body weights, body weight gain and food consumption were similar in all groups. Gravid uterus weights, carcass weights and net weight change from day 6 of gestation were similar in all groups. There were no maternal necropsy findings. Mean foetal body weights were similar in all groups and there were no treatment-related foetal external, visceral, or skeletal abnormalities. The results of this study suggest that the test material is considered to be non-toxic to dams and foetuses at doses up to 1000 mg/kg. There was no evidence for embryotoxic or teratogenic potential. The no observed effect level (NOEL) for rat dams and foetuses in this study was 1000 mg/kg body weight/day.

 

Khalil (1996b) investigated the potential of the test substance to cause embryotoxic, foetotoxic, and teratogenic effects in rabbits. The study was performed in accordance with standardised guidelines OECD 414, EC OJ No L133/24, Japanese MAFF 59 and US EPA OPP 83 -3, and under GLP conditions. The test substance was evaluated over a range of concentrations - 0, 10, 100 and 1000 mg/kg. Females were inseminated with semen from males of the same stock. Inseminated females were treated daily with the test material from day 7 to day 19 of gestation. On day 29 dams were sacrificed and foetuses extracted and examined for skeletal and visceral effects. None of the maternal animals died during the study and only occasional incidental clinical signs occurred during the study. At 1000 mg/kg, there was a slight reduction in food consumption during the dosing period (days 7 to 20) and an increase in food consumption at the end of gestation when the animals were no longer dosed (days 20 to 29). In this group a significant difference in weight change was seen only for days 12 to 16 of gestation and overall on days 7 to 19. Overall weight change for days 7 to 29 was comparable among all groups. Gravid uterus weights, carcass weights, and net weight change from day 7 were similar in all four groups. There were no maternal necropsy findings. Preimplantation losses, numbers of implantation sites and live litter size were similar in all groups. At 1000 mg/kg, there was an increase in the incidence of post implantation loss, almost exclusively in the form of early resorptions. Three females in this group showed total early embryonic resorption; for the remaining 16 pregnant females, this parameter was increased when compared to controls and historical control data. At 1000 mg/kg, foetal weights (both male and female) were slightly reduced compared to controls. However, the incidence and type of external, visceral and skeletal findings were not affected by treatment. Under the conditions of the study, maternal toxicity (reduced food consumption and body weight gain) and embryo and foetal toxicity (increased incidence of early resorption and decreased foetal weights) were seen in the 1000 mg/kg group. The No Observed Effect Level (NOEL) of the test material in this study was therefore considered to be 100 mg/kg for dams and foetuses. There was no indication of teratogenic potential.

In the study reported by FitzGerald (1993a), the teratogenicity of the test material was investigated in accordance with standardised guidelines OECD 414, EPA OPP 83 -3 , EC OJ No L133/24 and Japanese MAFF 59 under GLP conditions. During the study, the test material was administered to pregnant rats, daily, by gavage, from day 6 to day 15of gestation, at concentrations of 0, 50,. 250, 500 and 1000 mg/kg bw. Animals were checked daily for clinical signs and mortality; body weight and food consumption were measured daily/every 5 to six days, respectively. On day 21 of gestation dams were killed and subjected to gross necropsy. The uteri were dissected and contents examined. Live and dead foetuses were weighed, sexed and inspected for external abnormalities. They were then killed and placed in Bouin’s fixative. After at least four weeks fixation, all foetuses were subjected to visceral examination by microdissection.

Under the conditions of the study, none of the dams died and bodyweight, bodyweight gain and food consumption were unaffected by treatment. There were two cases of hair loss from day 15 on, in one animals dosed at 1000 mg/kg and one animal in the controls group. However, this was considered to be unrelated to treatment. Two dams in the 1000 mg/kg group had hemorrhagic perineal discharge (dam 33 on days 15 and 16 and dam 40 on day 14). Both of these dams had normal pregnancies and healthy litters (dam 33 had no resorptions and 13 live foetuses; dam 40 had one early resorption and 13 live foetuses. There were no other remarkable cage-side observations. Two animals were not pregnant: one in the 50 mg/kg group and one in the 500 mg/kg group. All other dams had viable foetuses. Pre- and post- implantation losses, number of live foetuses per litter and foetal weights were not affected by treatment. There were no dead foetuses. Mean gravid uterus and carcass weights, and net body weight gain from day 6 to day 21 did not differ significantly between groups. There were no remarkable observations in any animal at necropsy. 

There were no remarkable foetal external observations. Foetal visceral observations consisted of enlarged thymus and accessory liver lobulets, one case of haemothorax (250 mg/kg group), one ureteral fusion with renal pelvic dilation (500 mg/kg group), and renal pelvic dilation alone (1000 mg/kg group). None of these findings were considered to be related to treatment. There were no visceral malformations.

In conclusion the test material was not considered to be toxic to dams, embryos and foetuses at doses up to 1000 mg/kg; there was no indication of teratogenesis. The NOEL for maternal and developmental toxicity was therefore determined to be 1000 mg/kg bw/day.

 

In the study reported by FitzGerald (1993b), the teratogenicity of the test material was investigated in accordance with standardised guidelines OECD 414, EPA OPP 83 -3 , EC OJ No L133/24 and Japanese MAFF 59 under GLP conditions. During the study, the test material was administered to pregnant rabbits, daily, by gavage, from day 7 to day 19 of gestation, at concentrations of 0, 10, 100 and 1000 mg/kg bw. Animals were checked daily for clinical signs and mortality, body weight and food consumption were measured daily/every 5 to six days, respectively. On day 29 of gestation animals were killed and subjected to gross necropsy. The uteri were dissected and contents examined. Live and dead foetuses were weighed, sexed and inspected for external abnormalities. After killing and visceral examination by evisceration, foetal heads were separated from the trunks, fixed in Bouin's solution for at least three weeks and examined by a slicing technique.

Under the conditions of the study, there were no treatment-related clinical signs in pregnant rabbits. One of the animals in the 1000 mg/kg group had hair loss from day 16 on. Body weights were consistently higher in groups dosed at 10 and 100 mg/kg than in controls. These differences were not considered to be treatment-related. Body weight gain was not significantly affected by treatment, although the 100 and 1000 mg/kg groups had lower mean body weight gain than controls from days 16 to 20. Food consumption was reduced in the 1000 mg/kg group from day 16 to 20 (approximately half of control values). There were 5, 5, and 3 pregnant animals with viable foetuses at scheduled necropsy; one animal in the 100 and two in the 1000 mg/kg group were not pregnant, and one in the 100 mg/kg group had total resorptions. The incidence of postimplantation losses was not affected by treatment. Mean foetal body weights were slightly reduced in the 100 and 1000 mg/kg groups; in the 1000 mg/kg group, this could be attributable to the larger litter sizes; an effect on foetal body weight at 100 mg/kg might be apparent with larger sample size. Mean gravid uterus weight and net body weight change during pregnancy were not significantly affected by treatment. At schedules necropsy, one animal in the 100 mg/kg group and two in the 1000 mg/kg group had a bilateral uterus constriction; consequently these animals were not pregnant. There were no other remarkable findings.

Foetal external and visceral examinations (including head slicing) revealed no remarkable findings.

In conclusion, the test material was found to be slightly toxic to pregnant rabbits at 1000 mg/kg (reduced food consumption). However, there were no clear signs of foetal toxicity and no indication of teratogenesis during the study. The developmental NOEL was therefore determined to be 1000 mg/kg bw/day.

All of the above available data are deemed to be relevant, reliable and adequate for the purposes of risk assessment. An NOEL of 100 mg/kg/day (maternal and developmental toxicity) has been taken forward for risk assessment purposes.


Justification for selection of Effect on developmental toxicity: via oral route:
The key studies were selected on the basis that they were the most robust. The other studies available for this endpoint were rangefinding studies, which did not include skeletal examinations. The two key studies were performed on the preferred rodent and non-rodent species for this test. As the study with rabbits provided a lower NOEL, this was therefore selected above the study on rats for risk assessment purposes.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Part 3.7, Regulation 1272/2008, the test material does not require classification for reproductive toxicity. There was no indication of reproductive toxicity or of embryotoxic, foetotoxic or teratogenic potential of the test material in any of the studies available for assessment of reproductive and developmental toxicity.

In accordance with criteria for classification as defined by Directive 2001/59/EC, Annex VI, Point 4.2.3, the test material does not require classification for toxicity to reproduction

Additional information

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