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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Read-across performed with structurally similar substance.

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity (parents) was considered to be 1000 mg/kg/day and the reproductive/developmental No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day, for both males and females.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 August 2012 to 1 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read-across performed with structurally similar substance.
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD (SD)
- Age at study initiation: On dispatch from the supplier, the males were approximately 5 - 6 weeks old and the females were approximately 4 - 6 weeks old. At initiation of dosing, the animals were approximately 8 - 10 weeks old.
- Weight at study initiation: On dispatch from the supplier, the males weighed 158 - 200 g and the females weighed 112 - 156 g. At initiation of dosing, the animals weighed 330 - 460 g for males and 192 - 269 g for females.
- Housing: Animals were housed in cages, suspended on a series of racks. Male and female cages were racked separately.
Animals were housed in polycarbonate cages with stainless steel grid tops and solid bottoms, with approximate dimensions of 61 x 43.5 x 24 cm. Cages were suspended on moveable racks and fitted with water bottles and integrated stainless steel food hoppers.
The animals were initially housed 2 or 3 per cage. A few days prior to pairing for mating, males were transferred to individual cages with a stainless steel grid insert. After mating, the males were re-housed with their original cage mates.
Mated females were transferred to individual solid bottom cages. White paper tissue was supplied as nesting material from Day 20 of gestation.
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded), supplied by Special Diets Services Limited, Witham, Essex, UK.was provided ad libitum.
- Water (e.g. ad libitum): Water taken from the public supply (Scottish Water, Edinburgh, UK) was available ad libitum.
- Acclimation period: 27 days.
- Source: Charles River Limited, Margate, Kent, UK.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): A minimum of 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): A 12 hour light/dark cycle was in operation.

IN-LIFE DATES:
From: 27 August 2012
To: 12 October 2012
Route of administration:
oral: gavage
Vehicle:
other: 0.5 % w/v Methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The dosing formulations were prepared weekly, stored at ambient temperature and dispensed daily. All formulations were used within the 8 day stability period that was established previously at the testing laboratory.
The dosing formulations were stirred for at least 30 minutes prior to and throughout dosing.

VEHICLE
- Concentration in vehicle: 10, 30 or 100 mg/mL for the 100, 300 and 1000 mg/kg/day dose levels, respectively.
- Dose volume: 10 mL/kg. The volume administered to each animal was determined on each day by the weight of that animal recorded immediately prior to dosing, except during late gestation; from Day 16 of gestation until when parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.
Details on mating procedure:
- M/F ratio per cage: Pairings were on a one male to one female basis.
- Length of cohabitation: A few days prior to the initiation of mating, the males were separated into individual grid bottom cages. Animals were paired in ascending numerical order within each group. Each female was transferred to the cage of its appropriate co group male near the end of the work day, where it remained until mating had occurred or 14 nights had elapsed.
- Proof of pregnancy: Vaginal lavages were taken daily early each morning from the day of pairing until mating occurred and the stage of oestrus observed in each lavage was recorded. The day of presence of sperm in such a lavage was designated Day 0 of gestation. The time taken for each female to show a positive mating sign was evaluated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYTICAL METHOD
Analyses were performed by ICP-Optical Emission Spectroscopy using a validated analytical procedure. Samples to be analysed were transferred at ambient temperature to the analytical laboratory.

CONCENTRATION AND HOMOGENEITY ANALYSIS
Duplicate 0.5 mL samples were collected from the top, middle and bottom (duplicate middle only for control) from each formulation at each sampling time point (control samples not collected at Weeks 5 and 6) and were sent to the analytical laboratory.
Additional 0.5 mL triplicate samples were collected from the top, middle and bottom (triplicate middle only from control) from each formulation at each sampling time point (control samples not collected at Weeks 5 and 6) and were retained as back-up samples.
The results of the sample concentration were considered acceptable if they were within ± 10% of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentration of ≤10% for each group.
Duration of treatment / exposure:
The males were dosed for 4 weeks, starting 2 weeks prior to mating. The females were dosed 2 weeks prior to mating, throughout mating, gestation and through to at least day 4 of lactation.
Several females were not dosed on their respective days of parturition due to the animals starting to give birth prior to the commencement of dosing on that day.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were agreed after a review of existing relevant toxicological data, including a 14 day dose range finding study conducted at the testing facility in which dose levels up to 1000 mg/kg/day produced no adverse reaction to treatment.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Moribundity checks on all animals were carried out early morning and as late as possible each day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pre-trial, all animals received a detailed clinical examination. All animals were examined for reaction to treatment at approximately hourly intervals up to 4 hours post dose on each day of dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweights were recorded one week prior to the start of treatment. From the start of treatment, the individual bodyweights were recorded daily.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating. After pairing, the female food consumption was measured over Days 0 - 7, 7 - 14 and 14 - 20 of gestation and Days 0 - 4 of lactation. Male food consumption did not recommence after pairing for mating.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OTHER: Ophthalmoscopic examinations were carried out, haematology, coagulation and clinical chemistry parameters were evaluated and full functional tests to investigate any neurotoxic effects of the test material were also conducted.
Oestrous cyclicity (parental animals):
As part of the mating procedure, vaginal lavages were taken daily early each morning from the day of pairing until mating occurred (for up to 14 days) and the stage of oestrus observed in each lavage was recorded.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations: testis and epididymis weight.
Litter observations:
F1 GENERATION
- Litter size and sex: The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation.
- Clinical observations: The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily. Where practicable, any pups that were found dead or killed during lactation were sexed and appropriately examined. All pups were externally normal and were discarded following examination.
- Bodyweights: Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation.

OBSERVATIONS ON FEMALES WITH LITTERS DURING LACTATION
The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which the first pups were born) was designated as Day 0 of lactation. The duration of gestation was calculated.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding. White paper tissue was supplied to each mother for incorporation in the nest which was replaced when it became soiled.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: The males were killed when mating was completed and the animals had been dosed for at least 4 weeks.
- Maternal animals: The females were killed between Day 5 and 7 of lactation. Animals were killed by exposure to carbon dioxide followed by exsanguination.

GROSS NECROPSY
- All adult animals were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system, all external surfaces and orifices, cranial cavity and external surfaces of the brain and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all females were examined.
The following organs were weighed at necropsy for all adult animals before sampling and preservation: brain, epididymis, adrenal gland, pituitary gland, prostate gland, thyroid gland, heart, kidney, liver, lung, ovary, spleen, testis, thymus and uterus.

HISTOPATHOLOGY
Representative samples of the following tissues were collected from all adult animals and preserved as appropriate: animal identification (microchip), aortic artery, bone marrow smear, bone marrow (femur and sternum), femur (bone), rib (bone), sternum (bone), brain, cervix, epididymis, eye, adrenal gland, harderian gland, lacrimal gland, mammary gland, parathyroid gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (caecum, colon and rectum), larynx, liver, lung, lymph node (mandibular and mesenteric), skeletal muscle, nasal cavity, optic nerve, sciatic nerve, oesophagus, ovary, oviduct, pancreas, pharynx, skin, small intestine (duodenum, ileum and jejunum), spinal cord, spleen, stomach, testis, thymus, tongue, trachea, ureter, urinary bladder, uterus and vagina.
Histopathological evaluation of all tissues were undertaken for the 5 selected males and females in the control and high dose groups (the same animals that were used for the laboratory investigations).
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were killed by intra-peritoneal injection of sodium pentobarbitone between Days 5 and 7 of lactation.

GROSS NECROPSY
- Where practicable, animals found dead or killed prematurely were sexed and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Surviving pups were also examined for externally visible abnormalities. All pups were then discarded.
Statistics:
Where required to assist with interpretation, tests were applied to determine the statistical significance of observed differences between control and groups receiving test material. Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Pairwise comparisons were only performed against the control group.

Bodyweight and food consumption data, haematology, coagulation, clinical chemistry and selected FOB and motor activity data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test; i.e., pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).

Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal bodyweight as the covariate.
Reproductive indices:
For each group:
Male fertility index = Number siring a litter / Number paired
Female fertility index = Number pregnant / Number paired
Gestation index = Number bearing live pups / Number pregnant
Offspring viability indices:
For each litter and group:
Birth index = Total number of pups born (live and dead) / Number of implantation scars
Live birth index = Number of pups live on Day 0 of lactation / Total number born (live and dead)
Viability index = Number of pups live on Day 4 of lactation / Number live on Day 0
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no clinical signs during the course of the study that were considered to be related to administration of the test material.
- Animal 60 (female, 100 mg/kg/day) was not dosed on Days 2 - 4 of lactation, prior to scheduled euthanasia on Day 5 of lactation, due to displaying signs of pale skin and eyes, walking on tiptoes, dark/red discharge from the vagina, weight loss, staining on fur (ventral abdomen), body hunched and piloerection.
- Animal 68 (female: 300 mg/kg/day) was not dosed on Day 1 of lactation due to displaying signs of pale skin and eyes, walking on tiptoes, staining on fur (ventral abdomen), abnormal vocalisation (in hand of technician) and body hunched. At Day 2 of lactation, the animal suffered a total litter loss, however these signs were no longer present and the animal was dosed Days 2 - 4 of lactation, prior to scheduled euthanasia on Day 5 of lactation.
- These signs were considered to be related to difficulties experienced by these animals during parturition and/or the early part of lactation, and were considered to be incidental given their low absolute incidence and the absence of similar signs in animals receiving 1000 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the course of this study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Bodyweight gains were similar between control animals and animals receiving the test material.
- Mean bodyweight gain was slightly higher than controls in females receiving 1000 mg/kg/day over gestation Day 0 - 20 (approximately 13% more than the control group mean); however this was considered to be incidental given the considerable inter-animal variation within both the control and 1000 mg/kg/day dose groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Food consumption was similar between control animals and animals receiving the test material.
- Mean food consumption was noted to be higher than the controls in all dose groups between Days 0 - 4 of lactation; however this was considered incidental and a consequence of a slightly low mean Control group value.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In females during lactation, mean haemoglobin concentration, red blood cell counts and haematocrit were slightly lower than concurrent control at all dose levels (as low as 88 % of control mean at 1000 mg/kg/day), with a dose response relationship apparent in mean haemoglobin and haematocrit values. In addition, mean red cell distribution width values and reticulocyte counts were slightly higher than the concurrent control in these animals (reticulocytes as high as 18 % more than the control mean at 1000 mg/kg/day), with a dose response relationship apparent in mean red cell distribution width values. Although the changes were small in magnitude, many of the mean values were outside of the historical background control range, including all of these parameters at the highest dose level of 1000 mg/kg/day.
These changes were not apparent in males receiving the test material.
All other apparent differences in haematological or coagulation parameters were considered to be incidental due to large intra-group variation and/or the absence of a clear dose-response relationship.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Mean lactate dehydrogenase activities were lower than the concurrent control in a dose dependent manner in males at Week 4 (82, 79 and 65 % of control mean, at 100, 300 and 1000 mg/kg/day, respectively), and in females during lactation (87, 78 and 63 % of control mean, at 100, 300 and 1000 mg/kg/day, respectively), attaining statistical significance in males receiving 300 and 1000 mg/kg/day. These values were lower than the historical background control range, at all dose levels in females and at 1000 mg/kg/day in males.
In females during lactation, mean phosphate levels were higher than the concurrent control at 300 and 1000 mg/kg/day (36 and 64 % higher than the control mean, respectively), attaining statistical significance and being higher than the historical background control range at 1000 mg/kg/day. Mean calcium levels were also very slightly high at 300 and 1000 mg/kg/day (5 and 8 % higher than the control mean, respectively), achieving statistical significance at 1000 mg/kg/day, however all groups (including the control) were above the historical background control range.
All other apparent differences in clinical chemistry parameters were considered to be incidental due to large intra-group variation and/or the absence of a clear dose response relationship.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neurotoxicity Clinical Observations:
There were no neurotoxicity clinical observations during the course of the study that were considered to be related to administration of the test material.

Detailed Functional Observations:
Detailed functional observations in animals receiving the test material were similar to control animals throughout the course of the study. Any slight intergroup differences were considered to be too minor in magnitude to be attributed to the test material.

Motor activity:
Motion activity values in animals receiving the test material were similar to control animals. Any slight intergroup differences were considered to be too minor and/or transient to be attributed to the test material.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- Minimal granulomas containing pigmented material were observed in the lungs of two animals given 1000 mg/kg/day (32 Male and 71 Female). This was considered to be some type of foreign inhaled material, presumably due to reflux of test material during the dosing procedure.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat on this type of study, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
- Thymic atrophy and gastritis in females were considered to be secondary to stress associated with parturition.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of live pups born per litter, were similar between control females and females receiving the test material. The data are summarised in Tables 1 and 2.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were seen on reproductive performance up to the maximum test concentrations.
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
- The nature and incidence of the observations recorded for the dams and their pups were similar between control females and females receiving the test material.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- The mean number of live pups born per litter was similar between control females and those receiving the test material.
- Litter survival was similar between litters derived from control females and litters derived from females receiving the test material.
- Total litter losses were suffered by 1/10 females at 100 mg/kg/day, and 1/10 females at 300 mg/kg/day versus none in the control females. Given the low absolute incidence and the absence of any litter losses at 1000 mg/kg/day, these litter losses were considered incidental.
The data is summarised in Tables 3 and 4.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- Litter weights and mean pup weights were similar between litters derived from control females and litters derived from females receiving the test material.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Critical effects observed:
no
Reproductive effects observed:
not specified

Table 1: Mating Performance and Fertility Indices

Number of Nights to Positive Mating Sign

Dose Level (mg/kg/day)

0

100

300

1000

Number of Animals (number not becoming pregnant)

1

2

3

4

0

6

1

3

1

5

2

2

1

2

4

3

1

5

2

2

No clear indication of mating

Median no. nights to positive mating sign

Number passing one oestrus

0

2

0

0

2

0

0

3

0

0

2

0

Number of males paired

Number of siring males

Male Fertility Index (%)

Number of females paired

Number pregnant

Female Fertility Index (%)

10

10

100

10

10

100

10

10

100

10

10

100

10

10

100

10

10

100

10

10

100

10

10

100

 

Table 2: Group Mean Duration of Gestation and Overall Litter Performance

 

Dose Level (mg/kg/day)

0

100

300

1000

Number Pregnant

10

10

10

10

Duration of Gestation (Days)

20

21

22

23

Mean Duration

 

1

5

4

0

21.3

 

0

5

5

0

21.5

 

0

3

6

1

21.8

 

0

3

6

1

21.8

Number of females producing a live litter

Gestation index as %

10

100

10

100

10

100

10

100

Mean number of corpora lutea sites* per pregnancy ± SD

17.6 ± 1.4

18.3 ± 2.1

17.7 ± 3.5

19.2 ± 3.9

Mean number of implant sites* per pregnancy ± SD

15.6 ± 1.3

16.3 ± 1.7

15.6 ± 2.3

15.1 ± 2.3

Mean total number of pups born* per litter ± SD

13.8 ± 1.7

14.2 ± 1.9

13.7 ± 2.7

13.5 ± 3.1

Mean number of live pups* per litter ± SD

Day 0 of lactation

Day 1 of lactation

Day 4 of lactation

 

13.8 ± 1.7

13.8 ± 1.7

13.5 ± 1.7

 

13.8 ± 1.5

13.6 ± 1.5

13.6 ± 1.5

 

13.7 ± 2.7

13.4 ± 2.7

13.3 ± 2.6

 

13.4 ± 3.2

13.3 ± 3.1

13.1 ± 3.1

Total no. males** on Day 1 of lactation (%)

Total no. females** on Day 1 of lactation (%)

57 (46)

 66 (54)

45 (42)

62 (58)

54 (50)

53 (50)

58 (44)

75 (56)

* Excludes litters where all pups died

** Excludes litters where all pups died, excludes litters with mis-counted/sexed pups

 

Table 3: Group Mean F1 Survival Indices

 

Dose Level (mg/kg/day)

0

100

300

1000

 

Birth Index

Mean Litter Index

Number losing >2 pups

Number of litters

89

1

10

87

3

10

86

5

10

89

2

10

 

Live Birth Index

Mean Litter Index (%)

Number losing >1 pup

Number of Litters

100

0

10

97

1

10

100

0

10

99

0

10

Viability Index Days 1 - 4

Mean Litter Index (%)

Number losing >3 pups

Number of Litters

98

0

10

89

1

10

88

1

10

98

0

10

 

Table 4: Group Mean Litter and Pup Weight (g) ± Standard Deviation

Day of Lactation

Dose Level (mg/kg/day)

0

100

300

1000

Litter

Day 1

Day 4

 

86 ± 7

123 ± 19

 

83 ± 7

125 ± 13

 

88 ± 15

128 ± 23

 

88 ± 16

125 ± 24

                                 Mean of Litter Mean Pup Weight

Males

Day 1

Day 4

 

6.6 ± 1.0

9.5 ± 1.8

 

6.5 ± 0.5

9.6 ± 1.1

 

6.9 ± 0.8

10.0 ± 1.5

 

7.0 ± 1.0

10.1 ± 1.6

Females

Day 1

Day 4

 

6.3 ± 0.9

9.0 ± 1.8

 

6.0 ± 0.2

9.0 ± 0.7

 

6.5 ± 0.7

9.6 ± 1.5

 

6.6 ± 0.9

9.5 ± 1.6

Conclusions:
Under the conditions of this study in Sprague-Dawley strain rats the No Observed Adverse Effect Level (NOAEL) for reproductive effects was considered to be 1000 mg/kg/day for both males and females.
Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422.

Three groups of 10 male and 10 female Sprague-Dawley rats of the Crl:CD(SD) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (0.5 % w/v methylcellulose) following the same dosing regimen as the treated animals and were used as controls.

The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca. 6 weeks of treatment). 

The following parameters and end points were evaluated: clinical signs, bodyweights, bodyweight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathological examinations, mating and pregnancy performance, fertility, maternal care and pup performance (litter survival and pup weights).

Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of live pups born per litter were similar between control and treated females. Litter survival, litter weights and mean pup weights were similar between litters derived from control and treated females.

In conclusion, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity (parents) was considered to be 1000 mg/kg/day and the reproductive/developmental No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day, for both males and females.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across performed with structurally similar substance.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were seen on reproductive performance up to the maximum test concentrations.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Key result
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One GLP study, conducted to standardised guidelines is available on a structurally similar substance. The quality of the database is good.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Read-across performed with structurally similar substance.

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Three groups of 10 male and 10 female Sprague-Dawley rats of the Crl:CD(SD) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (0.5 % w/v methylcellulose) following the same dosing regimen as the treated animals and were used as controls.

The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca.4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca. 6 weeks of treatment). 

The following parameters and end points were evaluated: clinical signs, bodyweights, bodyweight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathological examinations, mating and pregnancy performance, fertility, maternal care and pup performance (litter survival and pup weights).

Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of live pups born per litter were similar between control and treated females. Litter survival, litter weights and mean pup weights were similar between litters derived from control and treated females.

In conclusion, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity (parents) was considered to be 1000 mg/kg/day and the reproductive/developmental No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day, for both males and females.

Effects on developmental toxicity

Description of key information

Read-across performed with structurally similar substance.

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity (parents) was considered to be 1000 mg/kg/day and the reproductive/developmental No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day, for both males and females.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 August 2012 to 1 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read-across performed with structurally similar substance.
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD (SD)
- Age at study initiation: On dispatch from the supplier, the males were approximately 5 - 6 weeks old and the females were approximately 4 - 6 weeks old. At initiation of dosing, the animals were approximately 8 - 10 weeks old.
- Weight at study initiation: On dispatch from the supplier, the males weighed 158 - 200 g and the females weighed 112 - 156 g. At initiation of dosing, the animals weighed 330 - 460 g for males and 192 - 269 g for females.
- Housing: Animals were housed in cages, suspended on a series of racks. Male and female cages were racked separately.
Animals were housed in polycarbonate cages with stainless steel grid tops and solid bottoms, with approximate dimensions of 61 x 43.5 x 24 cm. Cages were suspended on moveable racks and fitted with water bottles and integrated stainless steel food hoppers.
The animals were initially housed 2 or 3 per cage. A few days prior to pairing for mating, males were transferred to individual cages with a stainless steel grid insert. After mating, the males were re-housed with their original cage mates.
Mated females were transferred to individual solid bottom cages. White paper tissue was supplied as nesting material from Day 20 of gestation.
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded), supplied by Special Diets Services Limited, Witham, Essex, UK.was provided ad libitum.
- Water (e.g. ad libitum): Water taken from the public supply (Scottish Water, Edinburgh, UK) was available ad libitum.
- Acclimation period: 27 days.
- Source: Charles River Limited, Margate, Kent, UK.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): A minimum of 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): A 12 hour light/dark cycle was in operation.

IN-LIFE DATES:
From: 27 August 2012
To: 12 October 2012
Route of administration:
oral: gavage
Vehicle:
other: 0.5 % w/v Methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The dosing formulations were prepared weekly, stored at ambient temperature and dispensed daily. All formulations were used within the 8 day stability period that was established previously at the testing laboratory.
The dosing formulations were stirred for at least 30 minutes prior to and throughout dosing.

VEHICLE
- 0.5 % w/v Methylcellulose
- Concentration in vehicle: 10, 30 or 100 mg/mL for the 100, 300 and 1000 mg/kg/day dose levels, respectively.
- Dose volume: 10 mL/kg. The volume administered to each animal was determined on each day by the weight of that animal recorded immediately prior to dosing, except during late gestation; from Day 16 of gestation until when parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYTICAL METHOD
Analyses were performed by ICP-Optical Emission Spectroscopy using a validated analytical procedure. Samples to be analysed were transferred at ambient temperature to the analytical laboratory.

CONCENTRATION AND HOMOGENEITY ANALYSIS
Duplicate 0.5 mL samples were collected from the top, middle and bottom (duplicate middle only for control) from each formulation at each sampling time point (control samples not collected at Weeks 5 and 6) and were sent to the analytical laboratory.
Additional 0.5 mL triplicate samples were collected from the top, middle and bottom (triplicate middle only from control) from each formulation at each sampling time point (control samples not collected at Weeks 5 and 6) and were retained as back-up samples.
The results of the sample concentration were considered acceptable if they were within ± 10% of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentration of ≤10% for each group.
Details on mating procedure:
- M/F ratio per cage: Pairings were on a one male to one female basis.
- Length of cohabitation: A few days prior to the initiation of mating, the males were separated into individual grid bottom cages. Animals were paired in ascending numerical order within each group. Each female was transferred to the cage of its appropriate co group male near the end of the work day, where it remained until mating had occurred or 14 nights had elapsed.
- Proof of pregnancy: Vaginal lavages were taken daily early each morning from the day of pairing until mating occurred and the stage of oestrus observed in each lavage was recorded. The day of presence of sperm in such a lavage was designated Day 0 of gestation. The time taken for each female to show a positive mating sign was evaluated.
Duration of treatment / exposure:
The males were dosed for 4 weeks, starting 2 weeks prior to mating. The females were dosed 2 weeks prior to mating, throughout mating, gestation and through to at least day 4 of lactation.
Several females were not dosed on their respective days of parturition due to the animals starting to give birth prior to the commencement of dosing on that day.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were agreed after a review of existing relevant toxicological data, including a 14 day dose range finding study conducted at the testing facility in which dose levels up to 1000 mg/kg/day produced no adverse reaction to treatment.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Moribundity checks on all animals were carried out early morning and as late as possible each day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pre-trial, all animals received a detailed clinical examination. All animals were examined for reaction to treatment at approximately hourly intervals up to 4 hours post dose on each day of dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweights were recorded one week prior to the start of treatment. From the start of treatment, the individual bodyweights were recorded daily.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating. After pairing, the female food consumption was measured over Days 0 - 7, 7 - 14 and 14 - 20 of gestation and Days 0 - 4 of lactation. Male food consumption did not recommence after pairing for mating.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OTHER: Ophthalmoscopic examinations were carried out, haematology, coagulation and clinical chemistry parameters were evaluated and full functional tests to investigate any neurotoxic effects of the test material were also conducted.
SACRIFICE
- Male animals: The males were killed when mating was completed and the animals had been dosed for at least 4 weeks.
- Maternal animals: The females were killed between Day 5 and 7 of lactation. Animals were killed by exposure to carbon dioxide followed by exsanguination.

GROSS NECROPSY
- All adult animals were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system, all external surfaces and orifices, cranial cavity and external surfaces of the brain and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all females were examined.
The following organs were weighed at necropsy for all adult animals before sampling and preservation: brain, epididymis, adrenal gland, pituitary gland, prostate gland, thyroid gland, heart, kidney, liver, lung, ovary, spleen, testis, thymus and uterus.

HISTOPATHOLOGY
Representative samples of the following tissues were collected from all adult animals and preserved as appropriate: animal identification (microchip), aortic artery, bone marrow smear, bone marrow (femur and sternum), femur (bone), rib (bone), sternum (bone), brain, cervix, epididymis, eye, adrenal gland, harderian gland, lacrimal gland, mammary gland, parathyroid gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (caecum, colon and rectum), larynx, liver, lung, lymph node (mandibular and mesenteric), skeletal muscle, nasal cavity, optic nerve, sciatic nerve, oesophagus, ovary, oviduct, pancreas, pharynx, skin, small intestine (duodenum, ileum and jejunum), spinal cord, spleen, stomach, testis, thymus, tongue, trachea, ureter, urinary bladder, uterus and vagina.
Histopathological evaluation of all tissues were undertaken for the 5 selected males and females in the control and high dose groups (the same animals that were used for the laboratory investigations).
Ovaries and uterine content:
As part of the mating procedure, vaginal lavages were taken daily early each morning from the day of pairing until mating occurred (for up to 14 days) and the stage of oestrus observed in each lavage was recorded.
Fetal examinations:
F1 GENERATION
- Litter size and sex: The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation.
- Clinical observations: The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily. Where practicable, any pups that were found dead or killed during lactation were sexed and appropriately examined. All pups were externally normal and were discarded following examination.
- Bodyweights: Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation.

OBSERVATIONS ON FEMALES WITH LITTERS DURING LACTATION
The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which the first pups were born) was designated as Day 0 of lactation. The duration of gestation was calculated.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding. White paper tissue was supplied to each mother for incorporation in the nest which was replaced when it became soiled.

SACRIFICE
- The F1 offspring were killed by intra-peritoneal injection of sodium pentobarbitone between Days 5 and 7 of lactation.

GROSS NECROPSY
- Where practicable, animals found dead or killed prematurely were sexed and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Surviving pups were also examined for externally visible abnormalities. All pups were then discarded.
Statistics:
Where required to assist with interpretation, tests were applied to determine the statistical significance of observed differences between control and groups receiving test material. Unless otherwise stated, all statistical tests were two-sided and performed at the 5 % significance level using in-house software. Pairwise comparisons were only performed against the control group.

Bodyweight and food consumption data, haematology, coagulation, clinical chemistry and selected FOB and motor activity data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test; i.e., pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).

Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal bodyweight as the covariate.
Indices:
For each group:
Male fertility index = Number siring a litter / Number paired
Female fertility index = Number pregnant / Number paired
Gestation index = Number bearing live pups / Number pregnant

For each litter and group:
Birth index = Total number of pups born (live and dead) / Number of implantation scars
Live birth index = Number of pups live on Day 0 of lactation / Total number born (live and dead)
Viability index = Number of pups live on Day 4 of lactation / Number live on Day 0
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no clinical signs during the course of the study that were considered to be related to administration of the test material.
- Animal 60 (female, 100 mg/kg/day) was not dosed on Days 2 - 4 of lactation, prior to scheduled euthanasia on Day 5 of lactation, due to displaying signs of pale skin and eyes, walking on tiptoes, dark/red discharge from the vagina, weight loss, staining on fur (ventral abdomen), body hunched and piloerection.
- Animal 68 (female: 300 mg/kg/day) was not dosed on Day 1 of lactation due to displaying signs of pale skin and eyes, walking on tiptoes, staining on fur (ventral abdomen), abnormal vocalisation (in hand of technician) and body hunched. At Day 2 of lactation, the animal suffered a total litter loss, however these signs were no longer present and the animal was dosed Days 2 - 4 of lactation, prior to scheduled euthanasia on Day 5 of lactation.
- These signs were considered to be related to difficulties experienced by these animals during parturition and/or the early part of lactation, and were considered to be incidental given their low absolute incidence and the absence of similar signs in animals receiving 1000 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the course of this study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Bodyweight gains were similar between control animals and animals receiving the test material.
- Mean bodyweight gain was slightly higher than controls in females receiving 1000 mg/kg/day over gestation Day 0 - 20 (approximately 13% more than the control group mean); however this was considered to be incidental given the considerable inter-animal variation within both the control and 1000 mg/kg/day dose groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Food consumption was similar between control animals and animals receiving the test material.
- Mean food consumption was noted to be higher than the controls in all dose groups between Days 0 - 4 of lactation; however this was considered incidental and a consequence of a slightly low mean Control group value.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ophthalmic findings that were considered to be related to administration of the test material.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In females during lactation, mean haemoglobin concentration, red blood cell counts and haematocrit were slightly lower than concurrent control at all dose levels (as low as 88% of control mean at 1000 mg/kg/day), with a dose response relationship apparent in mean haemoglobin and haematocrit values. In addition, mean red cell distribution width values and reticulocyte counts were slightly higher than the concurrent control in these animals (reticulocytes as high as 18% more than the control mean at 1000 mg/kg/day), with a dose response relationship apparent in mean red cell distribution width values. Although the changes were small in magnitude, many of the mean values were outside of the historical background control range, including all of these parameters at the highest dose level of 1000 mg/kg/day.
These changes were not apparent in males receiving the test material.
All other apparent differences in haematological or coagulation parameters were considered to be incidental due to large intra-group variation and/or the absence of a clear dose-response relationship.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Mean lactate dehydrogenase activities were lower than the concurrent control in a dose dependent manner in males at Week 4 (82, 79 and 65% of control mean, at 100, 300 and 1000 mg/kg/day, respectively), and in females during lactation (87, 78 and 63 % of control mean, at 100, 300 and 1000 mg/kg/day, respectively), attaining statistical significance in males receiving 300 and 1000 mg/kg/day. These values were lower than the historical background control range, at all dose levels in females and at 1000 mg/kg/day in males.
In females during lactation, mean phosphate levels were higher than the concurrent control at 300 and 1000 mg/kg/day (36 and 64% higher than the control mean, respectively), attaining statistical significance and being higher than the historical background control range at 1000 mg/kg/day. Mean calcium levels were also very slightly high at 300 and 1000 mg/kg/day (5 and 8% higher than the control mean, respectively), achieving statistical significance at 1000 mg/kg/day, however all groups (including the control) were above the historical background control range.
All other apparent differences in clinical chemistry parameters were considered to be incidental due to large intra-group variation and/or the absence of a clear dose response relationship.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neurotoxicity Clinical Observations:
There were no neurotoxicity clinical observations during the course of the study that were considered to be related to administration of the test material.

Detailed Functional Observations:
Detailed functional observations in animals receiving the test material were similar to control animals throughout the course of the study. Any slight intergroup differences were considered to be too minor in magnitude to be attributed to the test material.

Motor activity:
Motion activity values in animals receiving the test material were similar to control animals. Any slight intergroup differences were considered to be too minor and/or transient to be attributed to the test material.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- No test-material related organ weight changes were noted. There were isolate organ weight values that were different from their respective controls. There were, however, no patterns, trends or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and unrelated to administration of the test material.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- No test-material related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat on this type of study, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- Minimal granulomas containing pigmented material were observed in the lungs of two animals given 1000 mg/kg/day (32 Male and 71 Female). This was considered to be some type of foreign inhaled material, presumably due to reflux of test material during the dosing procedure.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat on this type of study, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
- Thymic atrophy and gastritis in females were considered to be secondary to stress associated with parturition.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
- The mean number of live pups born per litter was similar between control females and those receiving the test material.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
- The mean number of live pups born per litter was similar between control females and those receiving the test material.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Details on maternal toxic effects:
Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of live pups born per litter, were similar between control females and females receiving the test material. The data are summarised in Tables 1 and 2.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No treatment-related effects
Fetal body weight changes:
no effects observed
Description (incidence and severity):
- Litter weights and mean pup weights were similar between litters derived from control females and litters derived from females receiving the test material.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - Litter weights and mean pup weights were similar between litters derived from control females and litters derived from females receiving the test material.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
- The mean number of live pups born per litter was similar between control females and those receiving the test material.
- Total litter losses were suffered by 1/10 females at 100 mg/kg/day, and 1/10 females at 300 mg/kg/day versus none in the control females. Given the low absolute incidence and the absence of any litter losses at 1000 mg/kg/day, these litter losses were considered incidental.
The data is summarised in Tables 3 and 4.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
- Litter weights and mean pup weights were similar between litters derived from control females and litters derived from females receiving the test material.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
- Litter survival was similar between litters derived from control females and litters derived from females receiving the test material.
External malformations:
no effects observed
Description (incidence and severity):
- The nature and incidence of the observations recorded for dams and their pups were similar between control females and females receiving the test material.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects
Developmental effects observed:
no

Table 1: Mating Performance and Fertility Indices

Number of Nights to Positive Mating Sign

Dose Level (mg/kg/day)

0

100

300

1000

Number of Animals (number not becoming pregnant)

1

2

3

4

0

6

1

3

1

5

2

2

1

2

4

3

1

5

2

2

No clear indication of mating

Median no. nights to positive mating sign

Number passing one oestrus

0

2

0

0

2

0

0

3

0

0

2

0

Number of males paired

Number of siring males

Male Fertility Index (%)

Number of females paired

Number pregnant

Female Fertility Index (%)

10

10

100

10

10

100

10

10

100

10

10

100

10

10

100

10

10

100

10

10

100

10

10

100

 

Table 2: Group Mean Duration of Gestation and Overall Litter Performance

 

Dose Level (mg/kg/day)

0

100

300

1000

Number Pregnant

10

10

10

10

Duration of Gestation (Days)

20

21

22

23

Mean Duration

 

1

5

4

0

21.3

 

0

5

5

0

21.5

 

0

3

6

1

21.8

 

0

3

6

1

21.8

Number of females producing a live litter

Gestation index as %

10

100

10

100

10

100

10

100

Mean number of corpora lutea sites* per pregnancy ± SD

17.6 ± 1.4

18.3 ± 2.1

17.7 ± 3.5

19.2 ± 3.9

Mean number of implant sites* per pregnancy ± SD

15.6 ± 1.3

16.3 ± 1.7

15.6 ± 2.3

15.1 ± 2.3

Mean total number of pups born* per litter ± SD

13.8 ± 1.7

14.2 ± 1.9

13.7 ± 2.7

13.5 ± 3.1

Mean number of live pups* per litter ± SD

Day 0 of lactation

Day 1 of lactation

Day 4 of lactation

 

13.8 ± 1.7

13.8 ± 1.7

13.5 ± 1.7

 

13.8 ± 1.5

13.6 ± 1.5

13.6 ± 1.5

 

13.7 ± 2.7

13.4 ± 2.7

13.3 ± 2.6

 

13.4 ± 3.2

13.3 ± 3.1

13.1 ± 3.1

Total no. males** on Day 1 of lactation (%)

Total no. females** on Day 1 of lactation (%)

57 (46)

 66 (54)

45 (42)

62 (58)

54 (50)

53 (50)

58 (44)

75 (56)

* Excludes litters where all pups died

** Excludes litters where all pups died, excludes litters with mis-counted/sexed pups

 

Table 3: Group Mean F1 Survival Indices

 

Dose Level (mg/kg/day)

0

100

300

1000

 

Birth Index

Mean Litter Index

Number losing >2 pups

Number of litters

89

1

10

87

3

10

86

5

10

89

2

10

 

Live Birth Index

Mean Litter Index (%)

Number losing >1 pup

Number of Litters

100

0

10

97

1

10

100

0

10

99

0

10

Viability Index Days 1 - 4

Mean Litter Index (%)

Number losing >3 pups

Number of Litters

98

0

10

89

1

10

88

1

10

98

0

10

 

Table 4: Group Mean Litter and Pup Weight (g) ± Standard Deviation

Day of Lactation

Dose Level (mg/kg/day)

0

100

300

1000

Litter

Day 1

Day 4

 

86 ± 7

123 ± 19

 

83 ± 7

125 ± 13

 

88 ± 15

128 ± 23

 

88 ± 16

125 ± 24

                                 Mean of Litter Mean Pup Weight

Males

Day 1

Day 4

 

6.6 ± 1.0

9.5 ± 1.8

 

6.5 ± 0.5

9.6 ± 1.1

 

6.9 ± 0.8

10.0 ± 1.5

 

7.0 ± 1.0

10.1 ± 1.6

Females

Day 1

Day 4

 

6.3 ± 0.9

9.0 ± 1.8

 

6.0 ± 0.2

9.0 ± 0.7

 

6.5 ± 0.7

9.6 ± 1.5

 

6.6 ± 0.9

9.5 ± 1.6

Conclusions:
Under the conditions of this study in Sprague-Dawley strain rats the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental effects was considered to be 1000 mg/kg/day for both males and females.
Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422.

Three groups of 10 male and 10 female Sprague-Dawley rats of the Crl:CD(SD) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (0.5 % w/v methylcellulose) following the same dosing regimen as the treated animals and were used as controls.

The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca.4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca. 6 weeks of treatment). 

The following parameters and end points were evaluated: clinical signs, bodyweights, bodyweight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathological examinations, mating and pregnancy performance, fertility, maternal care and pup performance (litter survival and pup weights).

Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of live pups born per litter were similar between control and treated females. Litter survival, litter weights and mean pup weights were similar between litters derived from control and treated females.

In conclusion, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity (parents) was considered to be 1000 mg/kg/day and the reproductive/developmental No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day, for both males and females.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across performed with structurally similar substance.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No treatment-related effects
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed
Developmental effects observed:
no
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One GLP study, conducted to standardised guidelines is available on a structurally similar substance. The quality of the database is good.
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

Read-across performed with structurally similar substance.

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Three groups of 10 male and 10 female Sprague-Dawley rats of the Crl:CD(SD) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (0.5 % w/v methylcellulose) following the same dosing regimen as the treated animals and were used as controls.

The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca.4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca. 6 weeks of treatment). 

The following parameters and end points were evaluated: clinical signs, bodyweights, bodyweight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathological examinations, mating and pregnancy performance, fertility, maternal care and pup performance (litter survival and pup weights).

Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of live pups born per litter were similar between control and treated females. Litter survival, litter weights and mean pup weights were similar between litters derived from control and treated females.

In conclusion, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity (parents) was considered to be 1000 mg/kg/day and the reproductive/developmental No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day, for both males and females.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproductive and developmental toxicity.

Additional information