Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
The substance  when given at dietary levels up to 1.25%  to females 7 days prior to mating and through mating and gestation, did not affect gestation length, size of pups, total number of young per litter, or stillbirths per litter compared to control animals.  
Link to relevant study records
Reference
Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
1983
GLP compliance:
no
Remarks:
Prior to GLP requirements
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Brookline, Massachusetts
- Age at study initiation: (P) weanling rats, 2 weeks acclimation, 8 weeks on test diets before initial pairings at about 105 days of age; offspring that would be parents in next generations weaned at 21 days and fed on same diets as progenitors
- Fasting period before study: none
- Housing: 4 females or 3 males per cage if not in mating portion of study; 2 females and 1 male during mating segment; 1 pregnant female in individual nesting box during gestation;

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): as libitum
- Acclimation period: initial animals observed 2 weeks prior to assigning to test groups

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 76.2 +/- 2 degrees F
- Humidity (%): 40 to 60 per cent desired
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:
Route of administration:
oral: feed
Vehicle:
other: Corn oil used to carry test article into feed
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Special preparation of Rockland Mouse and Rat diet containing no more than 1.0% of fat and free of any antioxidant commonly added as stabilizer and preservative to edible fats was the base diet. For the control diet, 1 kg corn oil added to 19 kg of base diet, and the mixture stirred for 20 minutes. This yielded 20 kg of diet (control) containing not more than 6.0% fat, and no antioxidants other than what occured naturally. For test diets, appropriate amount of test article added to corn oil to make 1 kg and this mixture added to 19 kg of base diet.
- Storage temperature of food: base diet received fresh every 2 months and stored under refrigeration until stored

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil used as carrier to introduce test article into diet, and to supply an adequate nutritional fat for a rat diet, as the base food was restricted to no more than 1.0% fat to avoid fat stabilizing antioxidants
- Concentration in vehicle: 300, 1200, 2000, 10,000 or 60,000 mg test article added to corn oil up to 1 kg.

Details on mating procedure:
- M/F ratio per cage: 1 male, 2 females
- Length of cohabitation: until females showed notable weight gain, or 4 weeks
- After 4 weeks of unsuccessful pairing replacement of first male by another male with proven fertility. Unsuccessful males placed with females of established fertility.
- After successful mating each pregnant female was caged (how): Individual nest boxes
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Initial progenitors: 8 weeks prior to first mating, then throughout gestation, and second mating period and gestation
F1, F2, or F3: from weaning if selected to reproduce, then through mating and two gestations
Frequency of treatment:
Initial group: fed control or treated diets for 8 weeks prior to breeding. Thereafter, animals were on appropriate feed for their group from weaning.
Details on study schedule:
- Age at mating of the mated animals in the study:
Initial animals: 105 days of age, 8 weeks on test diets initially. If mated, allowed to deliver, pups nurse for 7 days, then after 1 week allowed a second mating. If neither female in cage obviously pregnant, a second male placed with females after 4 weeks; second mating as described before.
F1 and F2: initially mated at about 3 months of age, had been on test diets with dams while nursing, and then from weaning on. Second mating after pups allowed to nurse for 7 days, and then allowed to mate after 1 week.

Selection of parents from F1 and F2: Pups of 2nd mating were allowed to nurse for 3 weeks (weaning). At that point, the offspring were grouped by sex, and counted within each dosing group. Animals were reduced in number by random selection to 20 females and 10 males to continue as progenitors of the next generation.
Remarks:
Doses / Concentrations:
15 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
60 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
100 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
3000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
20 females, 10 males per dose group for each breeding generation
Control animals:
yes, plain diet
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Initial parents: At study initiation
Pups: at birth, 4th day for first breeding offspring of each group, at 7th day and weaning for second breeding group of each dose

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OTHER:
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 7 postpartum: in the second breeding offspring for each dose group
- If yes: 20 females and 10 males randomly selected; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: Those that died or were killed and examined at 4, 7, or 21 days of age
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, ]

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

HISTOLOGICAL EXAMINATION OF PUPS:
The pups of the thrid generation (F3a and F3b) had histological examination of the brain, heart, lungs, kidneys, liver, spleen, intestinal tract, and adrenal glands
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced
- 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 of all animals dying during the test.

Postmortem examinations (offspring):
SACRIFICE
-The first litter of each initial female was sacrificed after suckling for 7 days. The offspring of the second litter were allowed to nurse 3 weeks, and those not selected for parenting were sacrificed. Pups in the first litters of the F1 and F2 females were counted and weighted at 4 days of age; those of the F1 females were then killed and dams remated. Pups in both litters of the F2 females and those in the second litter of the F1 females wee reduced to 10 pups, and allowed to suclke at 21 days of age. Representatives of the F3 generations were killed at 21 days of age.
- The F3 offspring were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated below were weighed and prepared for microscopic examination. Tissues were fixed in neutral formalin, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin.
Brain, heart, lungs, liver, kidneys, spleen, intestinal tract and adrenal glands
Reproductive indices:
Fertility index: Number of pregnancies/number of matings x 100
Gestation Index: Number of litters with live pups/Number of pregnancies x 100
Live Birth Index: Number alive at birth/ total number delivered x 100 (Based on first observation of the litters)
Offspring viability indices:
Viability Index: Number of Rats alive on 4th Day/ Total number Delivered x 100 (Day 7 for F1 generations)
Lactation index: Number of pups alive on 21st day/ Number continued after Day 4 x 100
Overall mortality among progeny throuhout 21 days of nursing (in %)
Clinical signs:
no effects observed
Description (incidence and severity):
associated with test article administration
Reproductive performance:
no effects observed
Description (incidence and severity):
associated with test article administration
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): Occasional fatalities among procreating animals were attributable to pneumonia and in some instances to dystocia. Throughout 3 genearations of procreators there werre 13 deaths among 180 males and 360 female progenitors. Three of the 10 females died of dystocia. The presence of disease among the animals, including control animals, was indicated by occasional anorexia, loss of body weight and rales in the lungs. It was thought that a dry atmosphere in the initial animal rooms contributed to the illness.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Test article administration to procreating animals had no effect on fertility or on the extent to which they produced live offspring. Exceptional variation in fertility index and in gestation index were attributed to influence of extraneous disease and not related to test article. Fertility of the initial progenitors (including controls) was poor. Subsequent generations had satisfactory performance. The number of pregnanccies resulting from the matings showed no relationship to test article in diet. During the two periods of mating of the initial progenitors, more pregnancies occurred in treated females (3000 ppm in diet) than in controls. The least number of pregnancies in th e(P) rats was in the animals with 60 ppm in the diet. Among the first generation of offspring (F1) the fertility index was lowest in the group fed 3000 ppm, and highest (100%) in the animals fed 500 ppm in the diet. Among the second generation (F2) of progeny, fertility was slightly low in both periods of mating in animals at 15 ppm in the diet. It was also slightly low in the first period, but not the second period, of mating of the rats fed 100 ppm. As there was no consistent pattern of effect, the differences in fertility were not thought to be attributalbe to test article administration.
Of the 180 male progenitors in the whole test, only 7 failed to sire litters; five of those were inital progenitors (3 at 60 ppm, 2 at 500 ppm). One male of the F1 generation on the 3000 ppm diet and one male of the F2 generation on the 15 ppm level also failed to sire offspring. These failures were attributed to sporadic disease unrelated to the test article.
The gestation index was lowest during the second period of mating of the original progenitors in the control group. It was consistently high among females of the 500 and 3000 ppm diet groups. Of the 60 females of the 3 generations on test, there were no differences attributable to test article in the numbers that produced 0, 1, or 2 litters when the means were compared by chi-square.

Dose descriptor:
NOEC
Effect level:
3 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: Parental, F1, F2, F3 (migrated information)
Clinical signs:
no effects observed
Description (incidence and severity):
associated with test article
Mortality / viability:
no mortality observed
Description (incidence and severity):
associated with test article
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No remarkable effect on weights at birth, day 4 (7) or when weaned at 21 days
Gross pathological findings:
no effects observed
Description (incidence and severity):
No anatomic anomaly, structural abnormality in the 5400 progeny examined. No gross pathology found in F2 and F3 offspring
Histopathological findings:
no effects observed
Description (incidence and severity):
No histopathology in tieeuse of third generation offspring examined
VIABILITY (OFFSPRING): Probability of survival at birth, and during first days of life was generally poor among th efirst generatin progeny ranging from 45.9 to 73% without regard to test article in the diets of the parents. The viability index improved in the second generation ranging from 89.4 to 97.4%, and was normal in the third generation except for the offspring of parental group receiving 60 ppm test article in diet; viability in that group was 84.9%, The poor life expectancy of the progeny was attributed in every instance to the influence of extraneous disease, and not the test article.
Survivability during nursing period (lactation index) ranged from 47.3 to 91.9% in the second generation and from 66.5 to 94.6% in the third generation; differences were not attributable to test article administered to the lactating females.

CLINICAL SIGNS (OFFSPRING): No remarkable differences in clinical signs were seen in the offspring of treated procreators compared with control pups

BODY WEIGHT (OFFSPRING): Content of test article in the procreating animals had no remarkable effect on body weights of the offspring at birth, at 4 days of age, or when weaned at 21 days.


GROSS PATHOLOGY (OFFSPRING): No gross pathological changes were found in the F2 and F3 offspring.

HISTOPATHOLOGY (OFFSPRING): Histopathological exam of the brain, heart, lungs, kidneys, liver, spleen, intestinal tract and adrenal glands of the third generation of offspring (F3) revealed no pathological alterations.

Reproductive effects observed:
not specified

The Gestation Index of Successive Generations of Rats on Diets with and without Test Article (Table 1 )

Test article concentration in diet (ppm)

1stMating Period (%)

2ndMating Period

(%)

Average for Both Matings (%)

Initial Parents (P)

0

100.0

75.0

87.9

15

91.7

89.5

90.3

60

100.0

100.0

100.0

100

100.0

83.3

90.9

500

100.0

93.8

96.8

3000

100.0

93.8

97.0

Generation F1

0

100.0

100.0

100.0

15

100.0

94.7

97.4

60

94.7

100.0

97.4

100

100.0

94.1

97.2

500

100.0

100.0

100.0

3000

100.0

100.0

100.0

Generation F2

0

100.0

100.0

100.0

15

100.0

100.0

100.0

60

100.0

90.0

94.7

100

100.0

100.0

100.0

500

100.0

94.7

97.4

3000

100.0

94.4

97.3

Gestation Index:   Number of litters with live pups/Number of pregnancies x 100

Groupings of Females Fed Each Diet and the Number of Litters They Produced (Table2)

Test Article Concentration in the Diet (ppm)

Number of females which produced the indicated number of litters

   Number of Litters

0

1

2

0

2

8

50

15

5

11

44

60

6

14

40

100

3

14

43

500

2

10

48

3000

4

11

45

No Test Article

2

8

50

Test Article Diets

20

60

220

Diets with and without Test Article (Viability Index) (Table 3)

Test article concentration in diet (ppm)

1stMating Period (%)

2ndMating Period

(%)

Average for Both Matings (%)

Generation F1

0

81.3

61.6

73.1

15

83.7

36.5

55.7

60

81.9

64.9

73.1

100

68.3

20.8

45.9

500

83.3

29.4

56.0

3000

74.7

31.5

56.2

Generation F2

0

96.3

98.5

97.4

15

96.3

90.4

93.3

60

92.3

95.7

94.1

100

99.4

80.3

89.4

500

98.6

88.8

93.4

3000

98.9

92.8

96.2

Generation F3

0

96.4

91.5

93.8

15

97.0

96.4

96.7

60

88.4

81.5

84.9

100

96.5

98.0

97.3

500

98.9

92.6

95.6

3000

98.8

94.9

96.6

* Seventh Day for Generation F1  

Viability Index: Number of Rats alive on 4thDay/ Total number Delivered x 100

Percentage Number of Animals Alive at Birth (Live birth Index) (Table 4)

Test article concentration in diet (ppm)

1stMating Period (%)

2ndMating Period

(%)

Average for Both Matings (%)

Generation F1

0

93.6

81.2

87.4

15

96.3

80.7

88.5

60

98.1

98.2

98.2

100

94.6

84.6

89.6

500

98.5

91.2

94.8

3000

98.8

92.3

95.6

Generation F2

0

97.9

100.0

99.0

15

97.2

96.1

96.6

60

94.2

97.0

95.6

100

100.0

98.9

99.4

500

99.0

98.3

98.6

3000

100.0

100.0

100.0

Generation F3

0

99.5

98.6

99.0

15

99.5

100.0

99.8

60

97.0

97.6

97.3

100

100.0

100.0

100.0

500

99.5

99.0

99.2

3000

100.0

97.2

98.6

Live Birth Index: Number alive at birth/ total number delivered x 100

(Based on first observation of the litters)

Conclusions:
The test substance was administered in diet at 0, 15, 60, 100, 500, and 3000 ppm (nominal) to 3 successive generations of breeding rats. Over the course of the study, animals were affected by environmental conditions and extraneous disease that were unrelated to test article in the diet. However, it can be concluded from this study that the substance in the diet up to levels of 3000 ppm did not affect fertilityof rats, viability of offspring, or caused abnormalities in the offspring under the conditions of this 3-generation study
Executive summary:

Diets containing the test substance in the nominal amounts of 0, 15, 60, 100, 500, and 3000 ppm were fed to 3 successive generations of breeding rats. Over the course of the study, animals were affected by environmental conditions and extraneous disease that were unrelated to test article in the diet. However, the data warrant the following conclusions:

1. Occasional fatalities among the breeding animals were attributable to respiratory disease, and in some cases dystocia.

2. The content of test article in the diet had no effect on fertility of the breeding animals, or on the extent to which they produced living offspring. Exceptional variations in fertility index and in gestation index were attributed to the influence of extraneous disease unrelated to test article concentration in diet.

3. The probability of survival at birth and during the first days of life, the viability index, was generally poor among the first generation ranging from 45.9 to 73 per cent without regard to test article concentration in the diet fed to the parents. The viability index improved in the second generation ranging from 89.4 to 97.4% and was normal in the third generation except for the group fed a test article diet containing 60 ppm. At that concentration level, viability was 84.9%. The poor life expectancy of offspring in the various groups was attributed to extraneous factors and not test article concentration in the diet.

4. The survival during the time the pups were nursed (the lactation index) ranged from 47.3% to 91.9% in the second generation, and from 66.5 to 94.6% in the third generation. The variation was not attributed to test article concentration in the diet.

5. The content of test article in the diets of the breeding animals exerted no remarkable effect upon the body weights of the offspring at birth, at 4 days of age, or when weaned at 21 days of age.

6. No anatomical anomaly or structural abnormality was found among 5400 rats that were offspring of animals on diets containing test article.

7. No gross pathological changes were found in the F2 and F3 offspring. Histological examination of the brain, heart, lungs, kidneys, liver, spleen, intestinal tract, and adrenal glands of the third generation of offspring revealed no pathologic alterations. 
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
rat
Quality of whole database:
Although the study is from 1969 and some deficiencies including infections in the animals are noted, no indications of a test substance related impairment of fertility have been observed up to the highes dose tested: 3000 ppm (nominal) in the diet. The findings are corroborated by the repeated dose studies in rats and dogs with a duration of 90 days and two years in which no significant changes in the reproductive organs were observed.
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 reproduction-developmental screening test according to OECD 421 did not reveal any effects on fertility or embryonic development at the highest recommended dose of 1000 mg/kg bw. The NOAEL for both parental and reproductive toxicity was 1000 mg/kg bw, the highest dose tested.

A 3 generation study in rats receiving the substance in the diet at nominal concentrations up to 3000 mg/kg diet (ppm) did not reveal any test substance related effect on fertility. The finding is corroborated by the absence of significant effects on the reproductive organs in repeated dose toxicity studies of 3 months and 2 years duration in rats and dogs at comparable dose levels. Consequently based on the available data, the substance is not classified for reproductive toxicity / fertiltiy according regulation EC 1272/2008 and amendments.

In order to reinvestigate previous findings and to address certain specific concerns, an enhanced Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) is proposed. The proposed study would be substantially augmented with additional parameters in order to investigate any potential endocrine effects.


Short description of key information:
A 3 generation study in rats receiving the substance in the diet at nominal concentrations up to 3000 mg/kg diet (ppm) did not reveal any test substance related effect on fertility. The finding is corroborated by the absence of signficant effects on the reproductive organs in repeated dose toxicity studies of 3 months and 2 years duration in rats and dogs at comparable dose levels.

Justification for selection of Effect on fertility via oral route:
A 3-generation reproduction toxicity study in rats receiving the substance in their diet is available and the key study for this endpoint.

Effects on developmental toxicity

Description of key information
Two developmental toxicity studies, one in rats and one in rabbits are available. Groups of pregnant rats or rabbits received either 15 or 150 mg/kg bw  per day by gavage from day 6 to 15  and day of gestation respectively. Concurrent vehicle control animals were included. No test substance related effects on maternal toxicity and embyo fetal development were observed up to the highest dose tested. From the repeated dose studies it can be concluded that the administrerd dose was close to the maximum tolerated dose. The absence of developmental toxicity is also corroborated by the 3-generation study in rats. The studies in combination give sufficient confidence to conclude  that the substance is unlikely to cause adverse effects to the developing embryo or fetus.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
A reproduction-developmental screening test according to OECD 421 did not reveal any effects on fertility or embryonic development at the highest recommended dose of 1000 mg/kg bw. The NOAEL for both parental and reproductive toxicity was 1000 mg/kg bw, the highest dose tested.
Two developmental toxicity studies, one in rats and one in rabbits are available. The studies are following a previously accepted test guideline, but were performed in 1970. Only two dose levels were administered by oral gavage. The absence of developmental toxicity is also corroborated by the 3-generation study in rats. The studies in combination give sufficient confidence to conclude that the substance is unlikely to cause adverse effects to the developing embryo or fetus.
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

One screening study and two developmental toxicity studies, one in rats and one in rabbits are available. In the latter groups of pregnant rats or rabbits received either 15 or 150 mg/kg bw per day by gavage from day 6 to 15 and day 6 to 18 of gestation respectively. Concurrent vehicle control animals were included. No test substance related effects on maternal toxicity and embyo fetal development were observed up to the highest dose tested. From the repeated dose studies it can be concluded that the administrerd dose was close to the maximum tolerated dose. The absence of developmental toxicity is also corroborated by the 3-generation study in rats. The studies in combination give sufficient confidence to conclude that the substance is unlikely to cause adverse effects to the developing embryo or fetus.

Consequently, based on the available data the substance is not classidfied for toxicity to reproduction/ developmental toxicity according to regulation 1272/2008 and amendments.


Justification for selection of Effect on developmental toxicity: via oral route:
Weight of evidence from two developmental toxicity studies in rats and rabbits and a 3 generation study in rats.

Toxicity to reproduction: other studies

Additional information

When pregnant rats were given diets containing the substance at concentrations of 0.125 to 1.25% of the dry weight of the food, there were no effects on length of gestation, size of pups, or total number of young per litter. There was not an increase compared to control in numbers of stillborn pups in the litters. This study corroborates the findings of the 2 available teratogenicity studies in rats and rabbits.

Justification for classification or non-classification

Based on the weight of evidence from all available studies, a 3 -generation dietary study in rats, repeated dose dietary studies of 90 -day and 2 year duration in rats and dogs, developmental toxicity studies by gavage in rats and rabbits, and a dietary study in rats covering the prenatal and complete gestation and parturition period in rats, that consistently showed no substance related effects on fertility and/or embryo-fetal development up to the highest dose tested, no classification for reproductive endpoints, neither fertility no developmental toxicity is warranted according to regulation EC 1972/2008 and amendments.