Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1994-11-24 to 1996-05-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted under the regulations of GLP and the respective guideline. A read-across approach was used. For justification please refer to IUCLID section 13.
Qualifier:
according to
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Version / remarks:
November 1982
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
May 1983
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF 59 NohSan No 4200, January 1985
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Breeding laboratories, CH 4414-Füllinsdorf, Switzerland
- Age at study initiation: P: 8 wks; F1: 8 wks
- Weight at study initiation: weight range at study initiation ±20 % of the mean value for each sex
- Fasting period before study: no
- Housing: individual
- Diet: KLIBA powdered diet no. 32-343-4., ad libitum
- Water: tap water, ad libitum
- Acclimation period: 14 to 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 60 ± 15
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1995-10-13 To: 1996-05-20

BATCH HEALTH CHECK
During the accliamatization phase, blood was drawn for White Bolld Cell differential counts and cirus seology frim the rats allocated to the batch health check group (BHC). After the vlood collection, the BHC animals were sacrificed and necropsied. A sample of all macroscopic abnormalities and also lung, liver, kidney, spleen and heart was fixed in buffered formalin and examined histologically. Animals allocated to the study were examined by a veterinarien during the acclimatization period before beeing authorized for use in the study.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
in diet
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: A 5 % premix was made freshly each week. Final diets were prepared weekly by direct dilution of the premix, except for the 500 ppm level, which was made by dilution of the 8000 ppm level and mixed for at least 20 min.
- Mixing appropriate amounts of the powdered test substance with powdered diet (incl. a 10 minutes dispersion at the ca. 1:4 test substance:diet stage)
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: for a maximum of 21 days
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol: An interval of minimum 10 days (after last litters weaned) was inserted between the F1A- and F1B mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance stability and homogeneity in diet has previously been confirmed in SANDOZ Study No. 448R. During the study, samples of diets prepared for week 1 and then at 3-monthly intervals were analyzed for test substance content to check the accuracy of mixing. Additionally the homogeneity of the final diets prepared for week 1 were analyzed.

Diet samples were analyzed for the test item content using HPLC method developed, verified and adapted during the study. The account of the adaption of the analytical method was the low solubility of the test substance in the exraction solvent and an extension of the chromatographic separation.

Extraction method I:
Rat diet was extracted with methanole by ultrasonication for 15 minutes. After standing for 10 minutes to allow sedimentation of solids an aliquot of approx. 20 mL was filtered through paper filter. The clear solution was diluted with acetonitrile and analysed for the test item by HPLC-UV.

HPLC determination method I:
Column: ODS-Hypersil RP-18, particle size: 5 µm (LC6), 250 x 4.6 mm
Mobile phase: 0.5 % aqeous solution of trifluoroacetic acid / acetonitrile (1:1, v:v)
Gradient: isochratic
Flow rate: 1.0 mL/min
Volume injected: 100 - 500 µL
Wavelength: 239 nm

Extraction method II:
Rat diet was extracted with acetonitrile containing 5 % dimethylsulfxode by ultrasonication for 15 minutes and by vigorous shaking before and after ultrasonication. After standing for 5 minutes to allow sedimentation of solids an aliquot of approx. 3 mL was filtered through a 0.45 µm disposable filter. The clear solution was diluted with acetonitrile/5% demethylsulfoxide and analysed for the test item by HPLC-UV.

HPLC determination method II:
Column: Zorbax SB-C18, particle size: 4 µm (LC7), 150 x 2.1 mm
Mobile phase: Solvent A: acetonitrile; solvent B: 1 % aqueous solution of acetic acid (v:v)
Gradient: 0 min: A 25 %, B 75 % - 9 min: A 45 %, B 55 % - 11 min: A 60 %, B 40 %
Flow rate: 0.2 mL/min
Volume injected: 3 µL
Wavelength: 239 nm

The analytical results confirmed an acceptable homogeneity of the diet mixtures. The results of the reanalysis proved that diet samples can be stored for more than 9 months under deep-frozen conditions without any degradation.
The analytical results confirm that a correct dosage occurred at each dose level (range : -1.3 % at dose levels 500 ppm, +1.2 % at dose level 2000 ppm, and +5.6 % at dose level 8000 ppm). With the exception of one analtical result at week 0 ( over-dosage of +20.8 %) all diet concentrations lie well between the acceptable limits ( ± 20 % of nominal for single analytical results, ± 10 % of nominal for averaged concentrations over the whole dosage period).
From the results of the analytical work obtained in this study part it is concluded that the test substance was homogeneously distributed in the diet and that concentrations of the test substance in the diet ensured adequate exposure of the test system.
Duration of treatment / exposure:
The test substance was administered during a 70-day F0-generation premating period and also during the mating, gestation and lactation periods of the F1A- and F1B-generations.
Selected F1A-animals were treated from weaning for at least an 84-day premating period and also during the mating, gestation and lactation periods of the F2A-generation.
Frequency of treatment:
Continously via feed
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 15 weeks
Remarks:
Doses / Concentrations:
500, 2000, 8000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
30, 120, 500 mg/kg bw/day
Basis:
actual ingested
Male animals during pre-mating period.
Remarks:
Doses / Concentrations:
40, 170, 700 mg/kg bw/day
Basis:
actual ingested
Female animals during pre-mating period.
No. of animals per sex per dose:
26 animals per sex per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were selected on the basis of results obtained in a previously conducted reproduction pilot study.
- Rationale for animal assignment: random
Positive control:
not applicable
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily for mortility and ill health, except at weekends and holidays where they were only checked once daily, and once a week for a detailed clinical inspection

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed at weekly intervals throughout the study. Females was weighed weekly during the premating period and on days 0, 7, 14 and 20 post coitum. Dams which had littered were weighed on days 0, 7, 14 and 21 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was measured weekly at the same intervals as recording of the bodyweight, except during the mating period in which males and females had access to the same feeder. Relative food consumption ratios and intake of the test substance were calculated on a weekly basis.
Oestrous cyclicity (parental animals):
From all F0- and F1-females daily vaginal smears were taken during the last 20 days of the corresponding premating period and for at least 10 days before the second mating period (F1B).
Vaginal smears were also taken from all "sperm negative" females during the entire second mating period (F1B). Based on the unaffected fertility in all matings the estrous parameters were not investigated.
Sperm parameters (parental animals):
From all F0 and F1 adult males sperm motility were videorecorded at necropsy, slides for sperm morphology prepared and fixed and the left testis stored at about -70° C for sperm count analyses. Based on the unaffected fertility parameters in all matings the sperm parameters have not been investigated.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1 and F2] offspring:
The litters were examined for litter size, live birth, stillbirth and any gross anomalies. Litters were caged together with the dam until weaning on day 21 of lactation. Pups were sexed and weighed by sex on day 0, 4, 7, 14 and individually on day 21 of lactation. Litters were not standardized. The dams and pups were observed daily for survival and behavioral abnormalities in nesting and nursing.

GROSS EXAMINATION OF DEAD PUPS:
Dead young, except if excessively cannibalized or autolytical, were autopsied (F1A pups were completely preserved).
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals when not longer necessary for the assessment of reproductive effects.
- Maternal animals: All surviving animals after the last litter of each generation was weaned

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

ORGAN WEIGHTS
From all F0 and F1 parental animals the following organs were weighed:
Uterus (with cervix) ovaries (without oviducts), testes, single epididymis (total and cauda), seminal vesicles (with coagulating glands and their fluids), prostata, brain, liver, kidneys, adrenal glands spleen, and thymus.

HISTOPATHOLOGY
Histopathological examination of the following organs were restricted to all F0- and F1-parental animals from the control and high-dose group:
Vagina, uterus with cervix, ovaries with oviducts (minimum of 10 sections randomly selected from one completely sectioned ovary per female to quantify oocytes), testis, intact epididymis (longitudinal section), seminal vesicles, prostata, coagulating gland, pituitary, mammary gland liver and grossly abnormal tissue
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed on day 21 post partum.
- These animals were subjected to postmortem examinations (macroscopic and microscopic examination) as follows:
For the F1A generation the whole body was fixed. Special attention was paid to the organs of the reproductive system. The remaining pups were killed, necropsied and checked for possible defects.

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

ORGAN WEIGTHS
For all pups examined macroscopically (2 pups/litter) the following organs were weighed:
Ovaries (without oviducts), testes, brain, liver, kidneys, adrenal glands, spleen, and thymus.

HISTOPATHOLOGY
Based on the treatment unrelated necropsy findings no histopathology were performed for pups.
Statistics:
All parental and litter data were recorded on-line and processed by a VAX-computer based software package (PSA) , except necropsy observations which were recorded manually on paper. The necropsy data were transferred into a VAX-computer based software package and submitted to the Principal Investigator for histopathology. Food mixture preparations were recorded and processed by a PC based software package. Means, standard deviations and statistical analyses of the various data were calculated, where appropriate.

The following statistical tests were performed:
Parametric data: ANOVA followed by Dunnet's test. If ANOVA failed, the non-parametric test was used.
Non-Parametric data: Kruskal-Wallis be followed by Mann-Whitney-U.
Count data: Chi-square followed by Fisher's exact test.
Reproductive indices:
The following reproductive indices were calculated for each group:
- Mating Index: (number of females mated / number of females paired) x 100
- Fertility Index: (number of females pregnant / number of females mated) x 100
- Gestation Index: (number of females with liveborn / number of females pregnant) x 100
Offspring viability indices:
The following offspring viability indices were calculated for each group:
- Livebirth Index: (number of pups liveborn / number of pups delivered) x 100
- Viability Index: (number of pups surviving day 4 / number of pups liveborn) x 100
- Lactation Index: (number of pups surviving day 21 / number of pups surviving day 4) x 100
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no clinical signs that distinguished treated animals from controls. The few non-scheduled deaths of parental animals were randomly distributed among groups and not considered to be related to treatment

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Lower total body weight gains were recorded at 8000 ppm in F0 and F1 parental males, and in F0 and F1 parental females in pre-mating and pregnancy phases. Significantly reduced total body weight gains were also noted for F0 parental males at 2000 ppm. No effects were seen at 500 ppm.

The following statistically significant deviations from control mean in food consumption, expressed
in grams per kg body weight per day, were considered to be related to treatment:
- Higher food consumption in F0 males at 2000 and 8000 ppm, with a dose-related trend
- Higher food consumption in F1 males at 8000 ppm
- Higher food consumption in F0 females at 2000 and 8000 ppm during pregnancy phases, with a dose-related trend
- Higher food consumption in F1 females at 2000 and 8000 ppm during pre-mating and pregnancy phases, with a dose-related trend.

Maternal food consumption during the lactation phases did not distinguish treated animals from controls.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The mean parental test substance intake in relation to dietary test substance concentration was usually higher in animals at 8000 ppm than in animals at 500 or 2000 ppm, which reflects the generally higher food consumption values at 8000 ppm. For each exposure level, the mean test substance intake was highest during the pregnancy phases (approximately 3-fold higher than pre-mating levels and approximately 50% higher than lactation levels).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Based on the unaffected fertility in all matings the estrous parameters were not investigated.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Based on the unaffected fertility parameters in all matings the sperm parameters have not been investigated.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The following statistically significant deviation from control mean was considered to be related to treatment:
The pre-coital interval (time to mating) was significantly shorter at 8000 ppm in the second F0 mating period. No statistically significant deviations in pre-coital interval were observed in the first F0 and F1 mating period, and the finding could, therefore, be incidental.
Mating, fertility, and gestation indices and the duration of gestation did not distinguish treated animals from controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The following statistically significant deviations from control mean in parental animals were considered to be related to treatment:
The asolute and relative (to body and brain) weights of the seminal vesicles were higher in F1 parents at 2000 and 8000 ppm, with a dose-related trend. Higher relative seminal vesicles weight were also recorded in F0 parents at 8000 ppm. No morphological correlate was detected.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The incidence, type and severity of histopathologic lesions did not distinguish treated parental animals from controls.
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
2 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lower weight gain and increased food consumption
Remarks on result:
other: Generation: F0 & F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Lower weight gain and increased food consumption
Remarks on result:
other: Generation: F0 & F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
170 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Lower weight gain and increased food consumption
Remarks on result:
other: Generation: F0 & F1 (migrated information)
Dose descriptor:
NOEL
Remarks:
(developmental)
Effect level:
2 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lower weight gains and higher proportion of runts at 8000 ppm.
Remarks on result:
other: Generation: F1 & F2 (migrated information)
Dose descriptor:
NOEL
Remarks:
(developmental)
Effect level:
550 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lower weight gains and higher proportion of runts at 2500 mg/kg bw/day.
Remarks on result:
other: Generation: F1 & F2 (migrated information)
VIABILITY (OFFSPRING)
Lower live birth and viability indices were observed in the F2A generation at 8000 ppm. One dam at 8000 ppm delivered all her F2A pups stillborn. The total pre-perinatal loss was significantly higher in the F2A generation at 8000 ppm. The lactation index was not affected.
The significantly lower viability index in the F1B generation at 500 ppm was not considered to be related to treatment in the absence of similar findings at higher dose levels. Mean litter size and sex ratio did not distinguish treated animals from controls.

BODY WEIGHT (OFFSPRING)
F1A male and female offspring at 8000 ppm showed significantly reduced weight gains during the lactation period, but similar effects were not observed in the F1B and F2A generations. No effects on body weight development of the offspring were noted in any generation at 2000 and 500 ppm.

ORGAN WEIGHTS (OFFSPRING)
The following statistically significant deviations from control mean in the offspring can not be excluded as possible treatment - related effects:
- Lower absolute and relative (to body) weights of the thymus in F1B males at 8000 ppm
- Lower absolute weights of the kidneys in F1B females at 8000 ppm
- Lower relative (to body) weights of the kidneys in F1B and F2A females at 8000 ppm

GROSS PATHOLOGY (OFFSPRING)
The following statistically significant deviations from control mean in the offspring were considered to be related to treatment:
- A higher proportion of undersized animals (runt) in the F1A and F1B generation at 8000 ppm.
- A higher percentage of F2A offspring at 8000 ppm were found to have no milk in the stomach.
There were no other necropsy findings in the offspring that distinguished treated animals from controls.
Dose descriptor:
NOEL
Remarks:
(reproductive performance)
Generation:
F1a
Effect level:
2 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects on Live Birth Index, Viability Index and increased Pre-Perinatal Loss in the F2-Generation at 8000 ppm.
Dose descriptor:
NOEL
Remarks:
(reproductive performance)
Generation:
F1a
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Effects on Live Birth Index, Viability Index and increased Pre-Perinatal Loss in the F2-Generation at 550 mg/kg bw/day.
Dose descriptor:
NOEL
Remarks:
(reproductive performance)
Generation:
F1a
Effect level:
170 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Effects on Live Birth Index, Viability Index and increased Pre-Perinatal Loss in the F2-Generation at 740 mg/kg bw/day.
Reproductive effects observed:
not specified

Table 1: Mean Body Weight Change (grams)

Sex

Study period

Generation

0 ppm

500 ppm

2000 ppm

8000 ppm

Males

week 0 -32

F0

296.5

288.7

272.1

242.9 **

week 0 -20

F1

230.5

233.4

217.1

199.8 **

day 0-21 p.p.

F1A

35.5

37.7

34.1

30.0 **

F1B

37.5

39.7

36.6

33.5

F2A

33.3

34.4

35.4

34.3

 

 

 

 

 

 

 

Females

Pre-mating

F0

79.5

80.7

74.4

65.5 **

F1

82.6

83.5

78.7

74.2 *

1st pregnancy

F0

106.5

100.0

103.6

91.6 **

F1

99.8

104.6

102.5

87.3 *

2nd pregnancy

F0

107.9

101.6

114.7

90.6 **

day 0-21 p.p.

F1A

34.7

35.9

33.4

29.8 **

F1B

36.0

39.4

35.8

32.5

F2A

32.9

33.8

35.5

33.8

*          p< 0.05

**         p< 0.01

 

Table 2: Mean Food Consumption (grams/kg bw/day)

Sex

Study period

Generation

0 ppm

500 ppm

2000 ppm

8000 ppm

Males

week 0-32

F0

54,6

54,7

56,5

58,3 *

week 0 -20

F1

61,2

62,1

63,2

69.0 **

 

 

 

 

 

 

 

Females

Pre-mating

F0

83.2

84.5

84.6

83.7

F1

82.4

84.0

87.9 *

92.7 **

1st pregnancy

F0

273.1

282.0

298.9 **

303.1 **

F1

257.9

264.5

290. 0 **

308.4 **

2nd pregnancy

F0

245.1

256.8

269.0 *

273.6 **

1st lactation

F0

196.4

186.6

207.2

203.9

F1

179.4

188.7

191.4

190.2

2nd lactation

F0

161.8

143.6

173.8

184.1

*          p< 0.05

**         p< 0.01

 

Table 3: Mean Test Substance Intake (mg/kg bw/day)

Sex

Study period

Generation

0 ppm

500 ppm

2000 ppm

8000 ppm

Males

week 0 -32

F0

 -

27,3

113,1

466,2

week 0 -20

F1

 -

31,1

126,4

552,2

 

 

 

 

 

 

 

Females

Pre-mating

F0

 -

42.2

169.2

669.6

F1

 -

42.0

175.9

742.0

1st pregnancy

F0

 -

141.0

597.7

2424.6

F1

 -

132.2

580.1

2467.3

2nd pregnancy

F0

 -

128.4

537.9

2188.7

1st lactation

F0

 -

93.3

414.4

1631.6

F1

 -

94.3

382.8

1521.6

2nd lactation

F0

 -

71.8

347.6

1472.4

 

Table 4: Litter data

Litter parameter

Generation

0 ppm

500 ppm

2000 ppm

8000 ppm

Mean Litter Size (total)

F1A

 11.7

 10.3

 11.8

 11.3

F1B

 11.0

 9.1

 11.6

 10.0

F2A

 11.2

 11.7

 11.0

 10.0

 

 

 

 

 

 

Sex Ratio (% males)

F1A

48.5

47.5

50.5

41.6

F1B

50.4

45.8

48.5

47.8

F2A

49.0

49.0

50.0

53.3

 

 

 

 

 

 

Live Birth Index (%)

F1A

96

96

98

97

F1B

97

95

94

99

F2A

98

99

98

93 *

 

 

 

 

 

 

Viability Index (%)

F1A

99

96

99

97

F1B

97

92

98

97

F2A

98

98

98

88

 

 

 

 

 

 

Lactation Index (%)

F1A

98

93

99

99

F1B

99

97

100

100

F2A

99

97

98

97

 

 

 

 

 

 

Pre-Perinatal Loss (%)

F2A

11

10

11

21 **

*          p< 0.05

**         p< 0.01

 

Table 5: Necropsy Observation

Litter parameter

Generation

0 ppm

500 ppm

2000 ppm

8000 ppm

Runt (%)

F1A

0.4

 2.8

0.9

8.0 **

F1B

0.0

 2.2

0.0

14.0 **

F2A

0.8

 1.4

0.0

 3.2

 

 

 

 

 

 

No milk in Stomach (%)

F1A

 4.0

 5.0

 2.6

 2.7

F1B

 1.9

 3.3

 3.3

 2.6

F2A

 1.2

 1.4

 2.8

8.4 **
5.2 % #

**         p< 0.01

#: excluding stillborn, no statistics performed

 

Table 6: Organ weights

Weight of thymus in males

Weight parameter

Generation

0 ppm

500 ppm

2000 ppm

8000 ppm

Absolute weight (g)

F1B

0.196

0.215

0.174

0.158 **

F2A

0.174

0.166

0.181

0.165

Relative to body weight

F1B

0.453

0.458

0.422

0.408 *

F2A

0.429

0.413

0.431

0.402

 

Weight of kidneys in females

Weight parameter

Generation

0 ppm

500 ppm

2000 ppm

8000 ppm

Absolute weight (g)

F1B

0.485

0.518

0.483

0.419 *

F2A

0.446

0.448

0.480

0.429

Relative to body weight

F1B

1.168

1.161

1.146

1.098 *

F2A

1.132

1.131

1.120

1.075 *

*          p< 0.05

**         p< 0.01

 

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
120 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The 2-Generation Study available is considered acceptable for the assessment of substance-induced effects on fertility and reproduction.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A Two Generation Study according to OECD TG 416 was conducted to assess the potential reproduction toxicity of the test substance in rats. A read-across to the corresponding acid was conducted. Groups of 26 male and 26 female Wistar rats (F0 generation) were exposed to the test substance at dietary concentrations of 0, 500, 2000 or 8000 ppm for 70 days and allowed to produce 2 litters (F1A and F1B). Selected pups (26 males and 26 females) from the F1A litters were weaned and in turn allowed to produce a litter (F2A), following an 84-day pre-mating period, under the designated treatment conditions.

All parental animals and offspring were checked daily for viability and signs of ill health. Detailed clinical inspections, food consumption and body weight determinations were performed weekly. Vaginal smears were taken daily on the last 20 and 10 days of F0 and F1 premating periods, respectively. The animals were mated (1 male & 1 female) for a maximum of 21 days, vaginal smears were examined daily for the presence of spermatozoa, and females were inspected daily for the appearance of a vaginal plug. Following parturition, the litters were examined for size, sex, live birth, stillbirth and any gross abnormalities. Selected offsprings were examined for vaginal opening and prepunital separation timepoints. Parental animals and offspring were killed on day 21 post partum or shortly thereafter and inspected for macroscopic abnormalities. Selected organs were weighed, and selected tissues were fixed. Microscopic examinations were performed on the following tissues of F0 and F1 parental animals at 0 or 8000 ppm: vagina, uterus with cervix, ovaries with oviducts, testis, epididymis, seminal vesicles, prostata, coagulating gland, pituitary, mammary gland, liver and grossly abnormal tissue.

 

The following findings were statistically significant and considered to be related to treatment at 8000 ppm:

There were treatment-related effects in both generations of parental animals.

Lower weight gains and a higher food intake were observed in both sexes during the premating periods, and in dams during the pregnancy periods. Higher absolute and relative weights of the seminal vesicles were recorded in F1 parental males at necropsy, but no microscopic correlate was found.

There were effects on live birth, viability and development of offspring.

Lower weight gains were observed in the F1A generation during the lactation period, and F1A and F1B generations had higher proportions of runt at necropsy on day 21 post-partum; in addition, lower absolute and relative weights of kidneys (females) and the thymus (males) were recorded in the F1B generation. Higher pre-perinatal losses and lower live birth and viability indices were observed in the second F2A generation, and a higher proportion of the F2A generation was found to have no milk in the stomach at necropsy on day 21 post-partum.

 

The following findings were statistically significant and considered to be related to treatment at 2000 ppm:

There were treatment-related effects in parental animals.

Lower total weight gains were recorded in F0 males, and a higher mean overall food intake was observed in F0 males, in F1 females during the pre-mating phase, and in F0 and F1 dams during pregnancy periods. Higher absolute and relative weights of the seminal vesicles were recorded in F1 parental males at necropsy but again no microscopic correlate was found.

There were no adverse effects on reproductive performance or the development of offspring.

 

No statistically significant deviation from control mean was noted for any parameter at 500 ppm.

 

The results of this study showed that a dose level of 8000 ppm caused adverse effects on reproductive performance and the development of the offspring.

At 2000 ppm, although treatment-related effects were observed in parental animals, there were no adverse effects on reproductive performance or the development of the offspring. Accordingly, this dose level can be regarded as the “no-adverse-effect-level” (NOAEL) for systemic toxicity and the NOEL for reproductive performance and developmental effects.

Based on food consumption in the pre-mating period the NOAEL/NOEL of 2000 ppm correspond to about 120 mg/kg bw/day for male animals and 170 mg/kg bw/day for female animals.

 


Short description of key information:
In a Two-Generation Reproduction Study the test substance was administered to 26 Wistar rats/sex/dose in diet at dose levels of 0, 500, 2000, 8000 ppm. Treatment-related effects in both generations of parental animals were found in the high dose group as well as effects on live birth , viability and developmental toxicity. Due to treatment-related effects on body weight and food consumption the NOAEL for systemic effects were determined to be 2000 ppm. The NOEL for reproductive performance and developmental toxicity was determined to be 2000 ppm (approx. 120 mg/kg bw/day for males and 170 mg/kg bw/day for females based on food consumption).
This study is acceptable and satisfies the guideline requirement for a 2-Generation Reproductive Study (OPPTS 870.3800 and OECD 416) in rats.

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

Effects on developmental toxicity

Description of key information
In a developmental toxicity study the test substance was administered to 25 female Wistar rats/dose by gavage at dose levels of 100, 300 and 1000 mg/kg bw/day from days 6 through 15 of gestation. Reduced maternal body weight gain and absolute and relative feed consumption values were observed in the 1000 mg/kg bw/day group. On the basis of these data, the NOAEL (maternal toxicity) of the test substance is 300 mg/kg/day. Reduced fetal body weights and reversible delays in sternal and caudal vertebral ossification were found in fetuses of the 1000 mg/kg bw/day. Therefore, the developmental NOAEL was determined to be 300 mg/kg bw/day.
In a developmental toxicity study the test substance was administered to 20 female New Zealand rabbits/dose by gavage at dose levels of 30, 100 and 300 mg/kg bw/day from days 6 through 19 of gestation. Based on adverse effects on the dams (weight loss, abnormal feces, death and abortions) observed in the high and mid dose group the NOAEL (maternal) was set at 30 mg/kg bw/day. The developmental NOAEL for the test substance was determined to be 100 mg/kg/day.
The developmental toxicity studies in the rat and the rabbit are classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700 and OECD 414) in rat and rabbit.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1994-02-09 to 1994-09-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted under the regulations of GLP and the respective guideline. A read-across approach was used. For justification please refer to IUCLID Section 13.
Qualifier:
according to
Guideline:
other: U.S. Environmental Protection Agency Pesticide Assessment Guidelines Subdivision F, 83-3
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
treatment from GD 6 to GD 15
Qualifier:
equivalent or similar to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
treatment from GD 6 to GD 15
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HRP, Inc., Denver, Pennsylvania
- Age at study initiation: 11 month
- Weight at study initiation: 2.8 to 5 kg
- Fasting period before study: no
- Housing: individual
- Diet: Certified Rabbit Diet #5322, ad libitum
- Water: R.O. water incl. chlorine, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 61-72
- Humidity (%): 40-60
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: Aqueous 0.5% methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Suspensions of the test substance were prepared daily

VEHICLE
- Justification for use and choice of vehicle: common vehicle
- Concentration in vehicle: 3, 10 and 30 mg/mL
- Amount of vehicle: 10 mL/kg
- Lot/batch no.: 922148
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method: HPLC-UV
Column: Supelcosil LC-18, 5µm 15 cm x 4.6 mm or equivalent
Mobile phase: ACN / 0.05 M trifluoroacetic acid in water (1/1, v/v)
Detector Wavelength: 280 nm
Injection Volume: 5 µL
Flow rate: 0.6 mL/min

Results of analyses of suspensions of the test substance demonstrated that homogeneous suspensions could be achieved for the range of dosages administered for this study. Concentrations prepared for the first and last day of dosage for this study averaged within +/- 10 % of the target value.
Details on mating procedure:
Timed-pregnant female rabbits were used.
Duration of treatment / exposure:
GD 6 to GD 19
Frequency of treatment:
Once a day
Duration of test:
24 days (up to GD 29)
Remarks:
Doses / Concentrations:
30,100, 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
20 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were selected on the basis of two dosage-range studies
- Rationale for animal assignment: random
- Route of administration rationale: The oral route (via gavage) was selected for use because- 1) in comparison with the dietary route, the exact dosage can be accurately administered and 2) it is one possible route of human exposure.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice each day
- Cage side observations: viability.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before and one hour after dosage from GD6 to GD19 and once from GD20 to GD29

BODY WEIGHT: Yes
- Time schedule for examinations: daily during dosage period and on GD20, GD24 and GD29

FOOD CONSUMPTION: Yes
- Time schedule for examinations: daily during dosage and post-dosage period

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: tissues with gross lesions
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
- Head examinations: Yes
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
Statistics:
I. Parametric:
Bartlett's Test - Analysis of Variance - Dunnet's Test

II: Nonparametric:
A: Kruskal-Wallis Test (<75% ties) - Dunn's Test
B: Fisher's Exact Test

III. Test for Proportion Data:
Variance Test for Homogeneity of the Binomial Distribution
Indices:
no data
Historical control data:
Historical control data from 105 studies are available.
Details on maternal toxic effects:
Details on maternal toxic effects:
The increase mortality and abortion incidences in Group IV were considered effects of the 300 mg/kg bw/day dosage of the test substance because these does generally had abnormal feces, persistent weight loss and reduced feed consumption from the beginning of the dosage period. Necropsy of five of these Group IV does that died or aborted revealed a gastric trichobezoar, an observation probably associated with altered gastric motality and emtying as the result of the high viscosity of the test substance suspension; a gastric trichobezoar also occurred in one Group IV doe that survived to scheduled sacrifice.

All other deaths and abortions were considered unrelated to the test substance because they were sequelae of intubation accidents, there were no observations indicative of effects of the test substance and the incidences were not dosage-dependent.
One death in each Group I, II, III and IV appeared to be sequelae of an intubation accident. One Group II doe that died did not have adverse clinical or necropsy observations or remarkable weight losses or reductions in feed consumption.

The premature delivery of a litter by one Group I doe on GD 29 was a spontaneous event unrelated to the vehicle.

One doe died on GD 28 during abortion of a litter of four late resorptions. Necropsy of the doe revealed a gastric trichobezoar interrelated with clinical observations, no weight gain and essentially absent feed consumption on GD 8 to 27.
One doe was found dead in GD 13. The litter consisted of nine embryos and two early resorptions. Although no adverse clinical or necropsy observation occurred in this doe, other characteristic effects of the test substance, weight loss and reduced feed consumption an GD 6 to 12 were present.
One doe was found dead on GD 28. The litter sonsisted of 11 apparently normal fetuses. Necropsy of the doe revealed a gestric trichobezoar interrelated with abnormal feaces, persistent weight loss after GD 13 and reduced feed consumption on GD 6 to 27.

Abnormal feces were characteristic effects of the test substance in Groups III and IV. Increased numbers of Group III and IV dies had soft or liquid feces or dried feces; significant (p<0.01) numbers of Group IV does had absent feces or mocoid feces.
All other clinical observations were considered unrelated to the test substance.

Significant numbers (p< 0.01) of Group IV does had a gastric trichobezoar, an observation presumed to be related to effects on feed consumption and gastric motility associated with the thick consistency of the high dosage suspension. All does that had a gastric trichobezoar, regardless of dosage group, had an interval of severly reduced feed consumption.

Group III and IV lost weight for the entire dosage period (GD 6 to 20). Average maternal body weights were comparable among the four doseage groups throughout the study.

Group IV absolute and relative feed consumption values for the entire dosage period (GD 6 to 20) were reduced to 78.8% and 76.5% of the Group I values, respectively. The severity of these effects tended to increase during the dosage period.
After completion of the dosage period, absolute feed consumption values tended to increase in all groups given the test substance (rel. feed consumption values were unaffected), observations interrelated with the weight gains that also occurred in these groups after completion of the dosage period.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Pregnancy occurred in 19, 16, 19 and 17 of the 20 naturally bred does in Group I through Group IV, respectively. 17, 15, 17 and 9 pregnant does in Groups I to IV, erspectively, were Caesarean-sectioned on GD 29. Caesarean-sectioning and litter parameters were unaffected by the test substance. The litter average for corpora lutea, implantations, litter sizes, live and dead fetuses, early and late resoptions, fetal body weight, percent male fetuses and percent dead or resorbed conceptuses were comparable among the four dosage groups.
Totals of 130, 111, 105 and 63 live, GD29 Caesarean-delivered fetuses in Group I through Group IV, respectively, were examined for gross external, soft tissue and skeletal alterations and average numbers of fetal ossification.
Combination of malformations and variations resulted in the following incidences for fetal alterations. In Groups I through IV, respectively, 14 (82.4%), 13 (92.8%), 12 (70.6%) and 8 (88.9%) litters had fetuses with any alterations observed. In these same respective dosage groups, the total numbers of fetuses with identified alterations were 45 (34.6%), 29 (26.1%) , 29 (27.6%) and 15 (23.8%).

Group IV had significant increases (P≤0.01) in the incidence of supernumerary thoracic ribs. This reversible fetal alteration is common at maternally toxic dosages, was evident as significant increases (P≤0.01) in the average numbers of pairs of ribs per fetus and related significant changes (P≤0.05) in the average numbers of thoracic and lumbar vertebrae (increases and decreases, respectively).
No other gross external, soft tissue or skeletal malformations or variations in the fetuses were considered effects of the test substance because: 1) the observations are frequent inthis strain of rabbit; 2) the incidences were within the ranges observed historically and/or 3) in the incidences were not dosage-dependent.
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
Prenatal developmental studies with rats and rabbits conducted under the regulations of GLP and according to guideline are available. The data base is considered sufficient for the assessment of developmental toxicity.
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

Developmental toxicity study on rats

Presumed pregnant Crl: CD®BR VAF/Plus® (Sprague-Dawley) female rats (25 per dosage group) were administered orally by gavage 0 (Vehicle, 0.5% methylcellulose) 100, 300 and 1000 mg/kg/day dosages of test substance on days 6 through 15 of presumed gestation. A read-across approach was conducted to the correspondig acid of the test substance.

Observations for viability were recorded at least twice each day of the study. General appearance was observed twice during acclimation and on day 0 of presumed gestation. The rats were also examined for clinical observations of effects of the test substance, abortions, premature deliveries and deaths immediately before and approximately one hour after intubation (days 6 through 15 of presumed gestation) and once daily during the postdosage period (days 16 through 20 of presumed gestation).

Body weights were recorded twice during acclimation. Body weights and feed consumption values were recorded on day 0 of presumed gestation and daily during the dosage and postdosage periods.

All rats were sacrificed by carbon dioxide asphyxiation on day 20 of presumed gestation, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. All rats were examined for the number and distribution of corpora lutea, implantations, early and late resorptions and live and dead fetuses. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations using a variation of the microdissection technique of Staples; the remaining fetuses were eviscerated, cleared, stained with alizarin red S and examined for skeletal alterations.

 

No deaths occurred. All clinical and necropsy observations were considered unrelated to the test substance.

Group IV had a transient reduction in body weight gain on days 6 to 9 of gestation, followed by body weight gains that were greater than or comparable to Group I values in the remainder of the dosage period. Group IV body weight gain for the entire dosage period (calculated as days 6 to 16 of gestation) was reduced to 95% of the Group I value, reflecting the transient reduction in body weight gain on days 6 to 9 of gestation. Body weight gain during the postdosage period (days 16 to 20 of gestation) was also reduced in Group IV.

Group IV had significantly reduced (P0.01) absolute (g/day) and relative (g/kg/day) feed consumption values on days 6 to 9 and 6 to 12 of gestation, observations interrelated with reduced body weight gain in this group.

Absolute feed consumption values in Group IV were also significantly reduced (P0.05) on days 9 to 12 and 6 to 16 of gestation.

 

Fetal body weights were reduced in Group IV; this effect was statistically significant (P0.05) in Group IV male fetuses. No other Caesarean-sectioning or litter parameters were affected by administration of the test substance to the dams at dosages as high as 1000 mg/kg/day.

Group IV had reversible delays in fetal sternal and caudal vertebral ossification [significant increases (P≤0.01) in the litter and fetal incidences of incompletely and not ossified sternal centers and a significant reduction (P≤0.01) in the litter average for ossified caudal vertebrae]. These variations were considered interrelated with the reduced fetal body weight in Group IV and resulted in the significant increase (P≤0.01) in the number of Group IV fetuses with any alteration. No other gross external, soft tissue or skeletal fetal alterations (malformations or variations) were caused by dosages of the test substance as high as 1000 mg/kg/day.

 

Conclusion

On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) of the test substance is 300 mg/kg/day (the 1000 mg/kg/day dosage reduced maternal body weight gain and absolute and relative feed consumption values). The developmental NOAEL is also 300 mg/kg/day (the 1000 mg/kg/day dosage group had reduced fetal body weights and reversible delays in sternal and caudal vertebral ossification).

 

Developmental toxicity study on rabbits

Timed-pregnant New Zealand White [HRA:(NZW)SPF] female rabbits (20 per dosage group) were orally by gavage administered 0(Vehicle, aqueous 0.5% methycellulose), 30, 100 and 300 mg/kg/day of test substance on day 6 through 19 of presumed gestation.

Observations for viability were recorded at least twice each day of the study. General appearance was observed on the day of arrival and before intubation on day 6 of presumed gestation. Additional examinations for clinical effects of the test substance, abortions, premature deliveries and deaths were conducted immediately before and approximately 60 minutes after intubation. These observations were also made once daily during the postintubation period (days 20 through 29 of presumed gestation).

Body weights were recorded once during the predosage period, daily throughout the dosage period and on days 20, 24 and 29 of presumed gestation. Feed consumption values were recorded daily throughout the study.

Rabbits that were found dead, aborted or were moribund sacrificed were necropsied and examined on the same day the event occurred. Pregnancy status and uterine contents were recorded. All surviving rabbits were sacrificed by intravenous administration of Beuthanasia®-D Special euthanasia solution on day 29 of presumed gestation and Caesarean-sectioned. The thoracic, abdominal and pelvic cavities of each rabbit were examined. The number and distribution of corpora lutea, implantations, early and late resorptions and live and dead fetuses was recorded.

Each fetus was examined for sex, visceral and skeletal alterations. Late resorptions were examined to the extent possible.

 

Group IV had increased mortality and abortion (P≤0.01) incidences, observations considered to be effects of the 300 mg/kg/day dosage of the test substance because these Group IV does generally had abnormal feces, persistent weight loss and reduced feed consumption from the beginning of the dosage period. Necropsy of five of these Group IV does that died or aborted revealed a gastric trichobezoar, an observation probably associated with altered gastric motility and emptying as the result of the high viscosity of the test substance suspension; a gastric trichobezoar also occurred in one Group IV doe that survived to scheduled sacrifice.

All other deaths and abortions were considered unrelated to the test substance because they were sequelae of intubation accidents, there were no observations indicative of effects of the test substance, and the incidences were not dosage-dependent. One Group I doe prematurely delivered a litter on GD 29.

Abnormal feces were characteristic effects of the test substances in Groups III and IV. Increased numbers of Group III and IV does had soft or liquid feces or dried feces; significant (P≤0.01) numbers of Group IV does had absent feces or mucoid feces. When statistical analyses are made excluding values for does that did not acclimate, one or more types of abnormal feces occurs in significant numbers (P≤0.05 to P≤0.01) of does in Groups III and IV. Two Group IV does had a red substance in the cage pan; one of these does was found dead on GD 20. The anal area of one Group IV doe that aborted was covered with fecal matter, an observation interrelated with abnormal feces (dried feces, no feces) in this doe.

Significant numbers (P≤0.01) of Group IV does had a gastric trichobezoar, an observation presumed to be related to effects on feed consumption and gastric motility associated with the thick consistency of the high dosage suspension. All does that had a gastric trichobezoar, regardless of dosage group, had an interval of severely reduced feed consumption.

Groups III and IV lost weight for the entire dosage period (calculated as GD 6 to 20). These effects of the 100 and 300 mg/kg/day dosages are more clearly evident when values for does that did not acclimate are excluded from analyses [weight losses for this Interval occur only in Groups III and IV, Group IV has significant weight loss (P≤0.05) on GD 12 to 15.] During the postdosage period (GD 20 to 29), Group IV gained the most weight; the value for GD 24 to 29 was significant (P≤0.01) when values for does that did not acclimate were excluded from analyses. This observation is considered to be a rebound phenomenon, as commonly occurs in these type of studies. Average maternal body weights were comparable among the four dosage groups throughout the study and did not significantly differ, regardless of inclusion or exclusion of the values for the does that did not acclimate.

Group IV absolute (g/day) and relative (g/kg/day) feed consumption values for the entire dosage period (calculated as GD 6 to 20) were reduced to 78.8% and 76.5% of the Group I values, respectively. Both parameters were significantly reduced (P≤0.05, values for all pregnant does) in Group IV on GD 7 to 8; the severity of these effects tended to increase during the dosage period. No other statistically significant differences occurred in these parameters regardless of inclusion or exclusion of the values for the does that did not acclimate.

After completion of the dosage period, absolute feed consumption values tended to increase in all groups given the test substance (relative feed consumption values were essentially unaffected), observations interrelated with the weight gains that also occurred in these groups after completion of the dosage period.

 

Caesarean-sectioning and litter parameters were unaffected by the test substance. The litter averages for corpora lutea, implantations, litter sizes, live and dead fetuses, early and late resorptions, fetal body weights, percent male fetuses and percent dead or resorbed conceptuses were comparable among the four dosage groups and did not significantly differ, regardless of whether values for does that did not acclimate were included or excluded in the statistical analyses.

Group IV had significant increases (P≤0.01) in the incidence of supernumerary thoracic ribs. This reversible fetal alteration is common at maternally toxic dosages, was evident as significant increases (P≤0.01) in the average numbers of pairs of ribs per fetus and related significant changes (P≤0.05) in the average numbers of thoracic and lumbar vertebrae (increases and decreases, respectively).

No other gross external, soft tissue or skeletal malformations or variations in the fetuses were considered effects of the test substance.

 

Conclusion

The maternal no-observable-adverse-effect level (NOAEL) for the test substance is 30 mg/kg/day. The 100 and 300 mg/kg/day dosages caused increased incidences of abnormal feces and weight loss for the entire dosage period; the 300 mg/kg/day dosage also caused deaths and abortions, gastric trichobezoars, reduced absolute (g/day) and relative (g/kg/day) feed consumption values for the entire dosage period, and increased weight gain after completion of the dosage period.

The developmental NOAEL for the test substance is 100 mg/kg/day. The 300 mg/kg/day dosage group fetuses had increased incidences of supernumerary thoracic ribs, a variation in fetal ossification that commonly occurs at maternally toxic dosages. There were no adverse effects on embryo-fetal viability, fetal body weight or sex, or fetal external or soft tissue morphology at dosages as high as 300 mg/kg/day. No fetal malformations were caused by dosages of the test substance as high as 300 mg/kg/day. A minor variation in skeletal development of the 300 mg/kg/day dosage group fetuses was the only developmental effect identified in this study. Thus, the test substance was not teratogenic to rabbit fetuses, even at the higher of two dosages (100 and 300 mg/kg/day) that were toxic to the does.

 


Justification for selection of Effect on developmental toxicity: via oral route:
This study was considered to be the most relevant as the rabbit was shown to be more sensitive than the rat regarding prenatal development toxicity of the test item.

Justification for classification or non-classification

Based on the available experimental data the test substance is not classified and labelled for reproduction/fertility and developmental toxicity according to Regulation (EC) No 1272/2008 (CLP) and Directive 67/548/EEC.