Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 May 2015 - 02 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Max temperature (exceeded on 1 occasion) and max humidity (exceeded on 17 occasions). It was considered that these deviations had no adverse impact on the scientific purpose of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored over silica gel and under nitrogen at low temperature.
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Stable for at least 16 days once formulated (in Arachis oil BP)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item formulated in Arachis oil BP
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Formulations prepared at 7.5, 25 and 87.5 (reduced to 62.5 at Day 9+) mg/mL
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as a homegenous liquid formulation.

OTHER SPECIFICS: No

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: ca. 12 weeks old
- Weight at study initiation: Males = 311 - 354 g and females = 177 - 227 g
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20 %
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 h:12 h (light: dark)

IN-LIFE DATES: 25 June 2015 (first day of treatment) - 08 August 2015 (final day of necropsy)

Experimental dates: 27 May 2015 - 11 July 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories U.K. Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least sixteen days. Formulations were therefore prepared fortnightly and stored at approximately 4 °C in the dark, under nitrogen and over silica gel.

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Concentration in vehicle: 7.5, 25, 87.5 (62.5 at Day 9+) mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): Not reported
- Purity: Not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulations investigated during the study were found to comprimise of the test item in the range of 91 - 101 % and thus the required content limit of ± 10 % with reference to the nominal content was met.

Three validity criteria to demonstrate the validity of the analytical method were met; specifity, linearity (r2 value of calibration cure = 0.998) and accuracy (recoveries = ± 10 % from fortified samples).
Details on mating procedure:
Mating

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Due to the early termination of Female No. 85, Male No. 73 was not paired with a female. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition

Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays.
The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Litter data

On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated
retrospectively from this data)

Physical development

All live offspring were assessed for surface righting reflex on Day 1 post partum.
Duration of treatment / exposure:
Up to 8 weeks
Frequency of treatment:
Daily
Duration of test:
Up to 8 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
Due to the excessive body weight loss/reduced food consumption during the first week of treatment and the early termination of one female on Day 9, the high dose level was reduced to 250 mg/kg bw/day from Day 9 onwards.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on resuts from a prelim test conducted at 50, 100, 250, 500 and 1000 mg/kg bw/day. The effects detected at 1000 and 500 mg/kg bw/day were sufficient to exclude these levels from consideration as high dose levels for the OECD 422 study. The effects at 250 mg/kg bw/day however were insufficient to exclude a dose level close to 250 mg/kg bw/day from further investigation.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: n/a
- Post-exposure recovery period in satellite groups: n/a

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). Additionally, observations were also made four hours following dosing (not at weekends).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and at weekly intervals thereafter. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION : Yes
- During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was measured daily during the pre-pairing phase of the study.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity.

LITTER OBSERVATIONS: Yes
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
Litter size, sex, viability, growth (body weight and gains), clinical signs and surface righting reflex.

GROSS EXAMINATION OF DEAD PUPS:
Yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Yes

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

POSTMORTEM EXAMINATION (OFFSPRING): Yes
SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Not examined

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (with cervix)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Not observed
- Number of late resorptions: Not observed
Fetal examinations:
- External examinations: No
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.

Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module.
Indices:
Reproductive and offspring viability indices calculated.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 350/250 mg/kg bw/day showed incidences of increased salivation from Day 4 until termination (males) and Day 27 (females).

The female that was killed in extremis on Day 9 also had hunched posture on Day 9.

No such effects were evident in animals of either sex treated with 100 or 30 mg/kg bw/day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female treated with 350 mg/kg bw/day was killed in extremis due to excessive body weight loss on Day 9. Microscopic examination of this female revealed evidence of inappetance (salivary gland inactive) and depletion in the bone marrow and lymphoid atrophy in the thymus along with metabolic changes in the liver. These are considered non-specific and an actual cause of death could not be established.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 350 mg/kg bw/day showed a statistically significant reduction in body weight gain during the first week of treatment. Actual body weight losses were evident in four males on Day 8. Following the reduction of the high dose level on Day 9, recovery was evident in these males during the next two weeks of treatment with a statistically significant increase evident during Week 3. However body weight gain was slightly reduced again during Weeks 4 and 5. Overall body weight gain for these males was 17% lower than controls.

Females treated with 350 mg/kg bw/day showed a statistically significant reduction in body weight gain during the first week of treatment. Actual body weight losses were evident in four females on Day 8. One female was killed in extremis on Day 9 due to excessive weight loss. This female had lost 17% of her initial body weight. Following the reduction of the high dose level on Day 9, some recovery was evident in these females during the second week of treatment however three females showed an actual body weight loss. No adverse effects were detected in females treated at 350/250 mg/kg bw/day during gestation or lactation.

No such effects were detected in animals of either sex treated with 100 or 30 mg/kg bw/day.

Females treated with 100 and 30 mg/kg bw/day showed a statistically significant increase in body weight gain during the first week of treatment. An increase in body weight gain is generally considered not to represent an adverse effect of treatment and the statistical significance attained at these dose levels is most likely due to the lower than expected control values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 350 mg/kg bw/day showed a reduction in food consumption during the first week of treatment. Following the reduction of the high dose level recovery was evident thereafter.

No such effects were detected in animals of either sex treated with 100 or 30 mg/kg bw/day or in any treated female during gestation or lactation.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 350 mg/kg bw/day showed a reduction in food conversion efficiency during the first week of treatment. Following the reduction of the high dose level recovery was evident thereafter.

Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males treated with 350/250 mg/kg bw/day showed an increase in overall water consumption during maturation. No such effects were detected in females treated with 350/250 mg/kg bw/day or in animals of either sex treated with 100 or 30 mg/kg bw/day.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters examined.

Females from all treatment groups showed a statistically significant reduction in activated partial thromboplastin time. The majority of individual values were within background control range and in the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance.

Males treated with 350/250 and 100 mg/kg bw/day showed a statistically significant reduction in monocytes. Males treated with 30 mg/kg bw/day showed a statistically significant reduction in total leukocyte count and lymphocytes. All of the individual values were within background control ranges and in the absence of a true dose related response the intergroup differences were considered not to be of toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined.

Males treated with 30 mg/kg bw/day showed a statistically significant reduction in phosphorus. All of the individual values were within background control range and in the absence of a similar effect at the high dose group the intergroup difference was considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the behavioural parameters at 30, 100 or 350/250 mg/kg bw/day.

There were no treatment related changes in functional performance considered to be related to treatment at 30, 100 or 350/250 mg/kg bw/day.

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 30, 100 or 350/250 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 350/250 mg/kg bw/day showed a statistically significant increase in adrenal weight both absolute and relative to terminal body weight.

No toxicologically significant effects were detected in females treated with 350/250 mg/kg bw/day or in animals of either sex treated with 100 or 30 mg/kg bw/day.

Males treated with 350/250 mg/kg bw/day showed a statistically significant increase in spleen weight both absolute and relative to terminal body weight. In the absence of any associated microscopic changes in this sex the intergroup difference was considered not to be an adverse effect of treatment.

Females from all treatment groups showed a statistically significant reduction in uterus weight both absolute and relative to terminal body weight. Males treated with 100 mg/kg bw/day showed a statistically significant increase in absolute and relative thyroid weight. In the absence of a true dose related response or any associated histopathological correlates in the uterus or thyroid the intergroup differences were considered not to be of toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The female that was killed in extremis had an enlarged gastro-intestinal tract at necropsy.

No toxicologically significant effects were detected in terminal kill animals of either sex treated with 350/250, 100 or 30 mg/kg bw/day.

One control female, one male and two females treated with 30 mg/kg bw/day, one female treated with 100 mg/kg bw/day and one male and two females treated with 350/250 mg/kg bw/day had reddened lungs at necropsy. One female treated with 100 mg/kg bw/day had a fluid filled left horn of the uterus. A fully formed feotus was also present in the left horn of the uterus in this female. In the absence of any histopathological correlates the intergroup differences were considered not to be of toxicological importance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following treatment-related microscopic abnormalities were detected:

Adrenals: cortical hypertrophy (zona fasciculata) was present at a minimal or mild level in animals of either sex from all treatment groups.

Spleen: increased hematopoiesis was present in females treated with 350/250 mg/kg bw/day. In two of these females an increase was also present in the bone marrow. A minor increase in hematopoiesis was present in females treated with 100 mg/kg bw/day. No such effects were detected in treated males or in females treated with 30 mg/kg bw/day.

Liver: centrilobular hepatocyte hypertrophy was present in three males treated with 350/250 mg/kg bw/day. No such effects were detected in treated females or in males treated with 100 or 30 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One control female and one female treated with 100 mg/kg bw/day that had a total litter loss had a gestation length of 24½ and 24 Days (respectively). It is not unusual for females with small litters and/or extended gestation periods to fail to maintain their litter. In the absence of any such effects at the high dose group, the intergroup difference was considered to be incidental.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Mating performance and fertility was unaffected by treatment and there were no treatment related effects detected in the offspring parameters observed.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: The reduced body weight gain/food consumption was considered to be the result of the administration of the higher dose level (350 mg/kg bw/day) and once the dose level had been reduced recovery was evident.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
not examined

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Not applicable
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Not applicable

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 4       Summary of incidence of mating performance, fertility and gestation lengths

Group

(mg/kg bw/day)

# of paired males

# of females

Pre-coital interval (days)

Mating index

(%)

Pregnancy index

(%)

Gestation length (days)

Females with live offspring

Parturition index (%)

Paired

Mated

Pregnant

1

2

3

4

22

22.5

23

23.5

24

24.5

Control

12

12

12

11

6

1

0

5

100

92

2

7

0

1

0

1

10

91

30

12

12

12

12

5

3

3

1

100

100

2

7

0

3

0

0

12

100

100

12

12

12

12

4

5

2

1

100

100

1

5

2

2

1

0

10

83

350/250

11

11

11

11

3

3

2

3

100

100

2

3

1

5

0

0

11

100

 

 Table 5      Group mean litter size and litter weights

Group

(mg/kg bw/day)

# of corpora lutea

# of implantation sites

Total # of offspring born

Number of live offspring

Litter weight

(g)

Offspring weight

(g)

Offspring body weight change

(g)

Day 1

Day 4

Day 1

Day 4

Day 1

Day 4

(Days 1- 4)

Males

Females

Males

Females

Males

Females

Control

11.6

11.2

9.6

9.3

9.3

55.59

78.80

6.21

5.85

8.85

8.44

2.63

2.58

30

11.7

11.5

10.7

10.7

10.3

60.76

82.91

5.96

5.71

8.35

8.15

2.39

2.44

100

11.2

11.1

9.8

9.7

9.7

59.46

83.33

6.29

6.01

8.78

8.52

2.49

2.51

350/250

12.2

11.7

9.8

9.5

9.4

56.85

80.77

6.16

5.88

8.82

8.83

2.66

2.95

 

Table 6      Group mean implantation losses and survival indices

Group

(mg/kg bw/day)

Pre-implantation loss

(%)

Post-implantation loss

(%)

Live birth index

(%)

Viability index

(%)

Control

4.1

12.6

97.0

100.0

30

1.7

7.3

100.0

97.4

100

0.8

12.2

98.9

100.0

350/250

3.3

17.5

95.5

98.4

 

Table 7      Offspring (F1) sex ratio values

Group

(mg/kg bw/day)

Sex ratio (% males) post partumday:

At birth

1

4

Control

53.1

51.5

51.5

30

46.3

46.3

46.4

100

65.0

64.6

64.6

350/250

51.2

46.6

46.8

 

Table 8      Offspring (F1) clinical signs

Observation

Group (mg/kg bw/day)

Control

30

100

350/250

Number of litters

11

12

11

11

Small

2F (1)

1M 4F (3)

1M (1)

1M 1F (2)

Cold

-

-

1M (1)

-

No milk in stomach

-

1M (1)

-

-

Total litter loss

1

-

1

-

Missing

-

3M 1F (1)

1U (1)

2M (2)

Found dead

8M 2F (3)

-

3M (2)

3M 1F (2)

Surface righting (% pass)

82.2

89.3

92.3

96.2

Applicant's summary and conclusion

Conclusions:
Mating performance and fertility was unaffected by treatment and there were no treatment related effects detected in the offspring parameters observed.

The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 250 mg/kg bw/day, the highest concentration tested.
Executive summary:

In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422) 1,4-Dihydroxy-2,2,6,6-tetramethyl-4-piperidinol was administered to 36 male and 36 female Wistar Han:RccHan:WIST strain rats by gavage at dose levels of 30, 100 and 350 (reduced to 250 at Day 9 onwards) mg/kg bw/day for up to 44 consecutive days. In addition, 12 male and 12 female control rats were dosed with vehicle (Arachis oil BP) alone.

 

A summary of adult responses to the test item are described below;

 

Mortality- One female treated with 350 mg/kg bw/day was killed in extremis due to excessive body weight loss on Day 9. Microscopic examination of this female revealed evidence of inappetance (salivary gland inactive) and depletion in the bone marrow and lymphoid atrophy in the thymus along with metabolic changes in the liver. These are considered non-specific and an actual cause of death could not be established. There were no further unscheduled deaths.

 

Clinical signs- Animals of either sex treated with 350/250 mg/kg bw/day showed incidences of increased salivation from Day 4. The female that was killed in extremis on Day 9 also had hunched posture on Day 9.

 

No such effects were evident in animals of either sex treated with 100 or 30 mg/kg bw/day.

 

Behavioural assessments- There were no treatment-related changes in the behavioural parameters at 30, 100 or 350/250 mg/kg bw/day.

 

Functional performance- There were no treatment related changes in functional performance considered to be related to treatment at 30, 100 or 350/250 mg/kg bw/day.

 

Sensory reactivity assessments- There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 30, 100 or 350/250 mg/kg bw/day.

 

Bodyweight- Animals of either sex treated with 350 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Actual body weight losses were evident in four males and four females on Day 8 and one female was killed in extremis on Day 9 due to excessive weight loss. Following the reduction of the high dose level on Day 9, recovery was evident in males during the next two weeks of treatment however body weight gain was reduced again during Weeks 4 and 5 and overall body weight gain for these males was lower than controls. Following the reduction of the high dose level some recovery was evident in females during the second week of treatment however three females showed an actual body weight loss. No adverse effects were detected in treated females during gestation or lactation.

 

No such effects were detected in animals of either sex treated with 100 or 30 mg/kg bw/day.

 

Food consumption and efficiency- Animals of either sex treated with 350 mg/kg bw/day showed a reduction in food consumption during the first week of treatment. Following the reduction of the high dose level recovery was evident thereafter.

 

No such effects were detected in animals of either sex treated with 100 or 30 mg/kg bw/day.

 

Water consumption- Males treated with 350/250 mg/kg bw/day showed an increase in overall water consumption during maturation. No such effects were detected in females treated with 350/250 mg/kg bw/day or in animals of either sex treated with 100 or 30 mg/kg bw/day.

 

Reproductive performance;

Mating- No treatment-related effects were detected in mating performance.

 

Fertility- No treatment-related effects were detected in fertility.

 

Gestation length- Gestation lengths were between 22 and 23ó days and the distribution of gestation lengths for treated females was essentially similar to control. One control female and one female treated with 100 mg/kg bw/day that had a total litter loss had a gestation length of 24ó and 24 Days (respectively).

 

Litter size, viability, development- Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum were comparable to controls.

 

Offspring body weight gain and litter weights at birth and subsequently on Days 1 and 4 post partum were comparable to control litters. Sex ratio and surface righting were also comparable to controls.

 

The clinical signs apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 30, 100 and 350/250 mg/kg bw/day.

 

Haematology- There were no toxicologically significant effects detected in the hematological parameters examined.

 

Blood Chemistry- There were no toxicologically significant effects detected in the blood chemical parameters examined.

 

Necropsy- The female that was killed in extremis had an enlarged gastro-intestinal tract at necropsy.

 

No toxicologically significant effects were detected in terminal kill animals of either sex treated with 350/250, 100 or 30 mg/kg bw/day.

 

Organ weights- Males treated with 350/250 mg/kg bw/day showed a statistically significant increase in adrenal weight both absolute and relative to terminal body weight. No toxicologically significant effects were detected in females treated with 350/250 mg/kg bw/day or in animals of either sex treated with 100 or 30 mg/kg bw/day.

 

Histopathological changes;

 

The following treatment-related microscopic abnormalities were detected:

 

Adrenals: Cortical hypertrophy (zona fasciculata) was present at a minimal or mild level in animals of either sex from all treatment groups.

 

Spleen: Increased hematopoiesis was present in females treated with 350/250 mg/kg bw/day. In two of these females an increase was also present in the bone marrow. A minor increase in hematopoiesis was present in females treated with 100 mg/kg bw/day. No such effects were detected in treated males or in females treated with 30 mg/kg bw/day.

 

Liver: Centrilobular hepatocyte hypertrophy was present in three males treated with 350/250 mg/kg bw/day. No such effects were detected in treated females or in males treated with 100 or 30 mg/kg bw/day.

 

In conclusion, a ‘No Observed Effect Level’ (NOEL) for systemic toxicity could not be established due to observed toxic effects at the lowest tested concentration (microscopic changes in the adrenals of both male and female rats). The reduced body weight gain/food consumption was considered to be the result of the administration of the higher dose level (350 mg/kg bw/day) and once the dose level had been reduced recovery was evident. All of the microscopic changes observed were considered to either be stress related or an adaptive change. For these reasons, the No Observed Adverse Effect Level (NOAEL) was considered to be 250 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 250 mg/kg bw/day.

 

This combined toxicity study with reproduction screening in the rat is acceptable and satisfies the guideline requirements for an OECD 422 in the rat.