Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
yes
Remarks:
Formulations were heated for 68 rather than 60 minutes. The lungs of female no. 56 were not fixed.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Waxy solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Lot 40265
- Expiration date of the lot/batch: 01 May, 2017
- Purity test date: 09 October, 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stability for at least 6 hours at room temperature was confirmed over the concentration range of 1 to 200 mg/mL test article in Arachis oil (vehicle).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was suspended in Arachis oil.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST: The test article was dosed suspended in Arachis oil.

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Den Bosch
- Age at study initiation: 10-14 weeks old
- Weight at study initiation: Female group means ranged from 207-209 grams.
- Fasting period before study: None
- Housing: Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Sterilized sawdust as bedding material and paper as cage-enrichment
- Diet (e.g. ad libitum): Pelleted roden diet (SM R/M-Z from SSNIFF Spezialdiaten GmbH, Soest, Germany), ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum.
- Acclimation period: At least 5 days prior to treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 C
- Humidity (%): 40-70%
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 February, 2016 To: 14 April, 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test article was suspended in Arachis oil.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was chosen based on trial formulations performed at Charles River Den Bosch and on information supplied by the Sponsor (3M Company).
- Concentration in vehicle: Concentrations were tested for stability in ranges from 1 to 200 mg/mL. Appropriate concentrations were utlilized to dose animals at 0 (control), 100, 300, and 600 mg/kg via oral gavage at a maximum dose volume of 5 mL/kg body weight.
- Amount of vehicle (if gavage): The maximum dose volume used was 5 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were analyzed by an Acquity UPLC system (Water, Milford, MA, USA).
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
From Days 6 to 20 post-coitum (14 doses).
Frequency of treatment:
Daily
Duration of test:
21 Days post-coitum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females were dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of a range-finding study.
- Rationale for animal assignment: Random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 2 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18, and 21 post-coitum.

FOOD CONSUMPTION : Yes
- Food consumption was measured on Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined:

OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Externally visible macroscopic fetal abnormalities were recorded.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Toxicologically relevant clinical signs were noted at 300 and 600 mg/kg. These included hunched posture (one female in each group), piloerection (two and three females, respectively), pale feces (one and six females, respectively) and lean appearance (two and sixe females, respectively).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (no. 56) at 300 mg/kg was killed in extremis on Day 16 post-coitum. Based on the findings at necropsy, the death was considered to have been caused by the gavage procedure and not related to treatment with the test article.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights were statistically significantly lower for females at 300 and 600 mg/kg than controls on Day 21 post-coitum. At 300 and 600 mg/kg, body weight loss was observed on Day 9 post-coitum and body weight gains were statistically significantly lower on Days 9-21 post-coitum. Body weight and body weight gain of females in the 100 mg/kg group remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption (absolute and relative) was statistically significantly lower at 300 and 600 mg/kg on Days 6-9 post-coitum. For the remainder of the treatment period, food consumption was similar to control values.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Emaciation noted for one female at 300 mg/kg and two females at 600 mg/kg confirmed the clinical sign of lean appearance observed during the in-life phase of these animals. All other macroscopic observations at necropsy were considered not treatment-related as single females were affected and no dose-response relationship was observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There were no treatement related effects on litter size for any group. Mean litter sizes were 10.9, 9.2, 9.3, and 9.8 fetuses/litter for the control, 100, 300, and 600 mg/kg groups, respectively.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on pre- and post-implantation loss by treatment up to 600 mg/kg.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No total litter losses by resorption were noted during the study.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on early or late resorptions noted in the study.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the number of dead fetuses in the study.
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
One female (no. 69) at 600 mg/kg had an early delivery of two pups on Day 21 post-coitum. There were ten remaining viable pups inside the uterus of this female. This early delivery was an isolated finding and was therefore not considered to be treatment-related.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): One female (no. 69) at 600 mg/kg had an early delivery of two pups on Day 21 post-coitum. There were ten remaining viable pups inside the uterus of this female. This early delivery was an isolated finding and was therefore not considered to be treatment-related.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the number of pregnant dams.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
clinical signs
body weight and weight gain
food efficiency
gross pathology

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 300 and 600 mg/kg resulted in statistically significantly lower fetal body weights in both sexes. Mean fetal body weights (sexes combined) were 5.2, 5.1, 4.6 and 4.3 grams for the control, 100, 300, and 600 mg/kg groups, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size for any group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 600 mg/kg. Mean sex ratios (males: females) were 49:51, 47:53, 53:47, and 53:47 for the control, 100, 300, and 600 mg/kg groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size for any group.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment up to 600
mg/kg.

Malformations were noted in two fetuses at the 600 mg/kg high dose level and in one control fetus. At the high dose level, two litter mates (A080-02 and -09) had cleft palate, both confirmed skeletally, and the control fetus (A001-01) had an omphalocele and absent eye bulges. The occurrence of two of the same malformations in one litter suggests a genetic origin, rather than another cause. Thus, despite its occurrence at the high dose level, it was not considered to be treatment related.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There was a dose related increase in the incidence of total skeletal variations in Groups 3 (300mg/kg) and 4 (600 mg/kg), reaching statistical significance in Groups 4. Incidences were 77.7%, 69.6%, 87.1% and 90.4% per litter in Groups 1, 2, 3 and 4, respectively. This was due to statistically significantly higher incidences for 14th full ribs, caudal shift of pelvic girdle and unossified metatarsals in Groups 3 and 4. The incidences for 14th full ribs were 5.7%, 9.9%, 19.5%, 28.4%, for caudal shift of pelvic girdle 5.0%, 13.2%, 29.8%, 45.0% and for unossified metatarsals 0.8%, 5.1%, 11.7% and 49.6% per litter in Groups 1, 2, 3 and 4, respectively. The incidences of these variations in Groups 3 and 4 were also (far) above their historical control maximum values (13.1%, 12.8% and 6.3% per litter for 14th full ribs, caudal shift of pelvic girdle and unossified metatarsals, respectively) and were therefore considered to be treatment related. It should be noted that the higher incidences of unossified metatarsals in Groups 3 and 4 were in line with the marked lower mean fetal body weights of these groups (4.6 and 4.3 grams respectively versus 5.2 grams in the control group) and thereby in line with delayed fetal development.

In Groups 3 (300 mg/kg) and 4 (600 mg/kg), the variation of 7th cervical ossification sites was not observed, whereas it was noted at 7.1% and 2.9% per litter in Groups 1 (0 mg/kg) and 2 (100 mg/kg), respectively, resulting in statistically significantly lower incidences in Groups 3 and 4. Because these sites of ossification disappear postnatally by incorporation in the transverse process of cervical vertebra no. 7, they can also be regarded as ossification parameter. By taking this into account together with the lower fetal body weights in Groups 3 and 4, the absence of 7th cervical ossification sites in Groups 3 and 4 is considered to be a sign of delayed fetal (skeletal) development and to be related to treatment.

Of the remaining skeletal variations, the finding of bent ribs showed a variable group distribution. Incidences were 19.1%, 4.8%, 14.5% and 2.3% in Groups 1, 2, 3 and 4, respectively, and statistical significance was reached for the lower incidences in Groups 2 and 4. The reason for this is unknown, but the group distribution did not indicate a treatment relationship. In addition, all incidences were within the historical control data range (0.8 - 22.3% per litter) and a lower incidence of this finding is not considered to be a detrimental effect. Therefore, the notable group distribution of bent ribs was considered to have occurred by chance.

The other skeletal variations that were noted occurred in the absence of a dose-related incidence trend, occurred infrequently or were observed in control fetuses only. Therefore, they were not considered to be treatment related.

Skeletal malformations were observed in 4 (4), 0 (0), 8 (6) and 2 (2) fetuses (litters) in dose Groups 1, 2, 3 and 4, respectively. The ones in Group 4 (600 mg/kg) had severely maligned sternebrae (A074-06) or a rib anomaly (A070-05) and the ones in Group 3 (300 mg/kg) either had a rib anomaly (A054-02 and -04), malpositioned metacarpals or metatarsals (A045-01 and A059-05), bent limb bones (A054-06, A062-02 and A063-01) or a vertebral anomaly with or without associated rib anomaly (A051-10). The latter two malformations also occurred in concurrent control fetuses (bent limb bones in fetus A007-01 and a vertebral anomaly with or without associated rib anomaly in fetuses A009-05 and A012-06). All these malformations were considered to be chance findings, because their single occurrence and/or group distribution did not indicate a relation to treatment.

The remaining skeletally malformed control fetus was the one with external malformations (A001-01) which appeared to have fused mandibles and sternoschisis as well.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 600 mg/kg.

Three viscerally malformed fetuses were revealed at fetal examination and all three were from the 600 mg/kg high dose group. Two fetuses (A067-08 and A083-10) had a small eye that was noted at serial sectioning of the head and in fetus A072-12 all internal organs were laterally transposed. It should be mentioned that control fetus A001-01 also had small (or absent) eyes, but this was revealed externally by absent eye bulges and confirmed skeletally by small orbits. Taking this into account, and the fact that in historical control fetuses absent and/or small eyes and situs inversus are the most common visceral malformations, the occurrence of both malformations in the high dose group was considered to have occurred by chance and was as such not related to treatment.
Visceral variations that were noted in the treated groups of this study were small supernumerary lobe(s) and appendix of the liver, discolored liver, partially undescended thymus horns, and convoluted and/or dilated ureters. These variations occurred at low incidences, in the absence of a dose-related incidence trend and/or were noted in control fetuses and therefore were not considered to be treatment related.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test article is 100 mg/kg.
Executive summary:

The prenatal developmental toxicity potential of the test article was evaluated in female Wistar Han rats. The study was performed in compliance with OECD GLP (1997). The test method was based on OECD 414 (2001), EPA OPPTS: 870.3700 (1998) and EC No 440/2008 Part B. There were no deviations that affected the outcome of the study or the interpretation of the results. Bred female Wistar Han rats (22/group) were dosed via oral gavage once daily with 0 (control), 100, 300, or 600 mg/kg test article in arachis oil from Days 6 to 20 post-coitum. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity. All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laprohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded and corrected body weights (changes) were calculated. The fetuses were weighed, sexed, and examined for external, visceral and skeletal malformation and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative; these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stain with Alizarin Red S for skeletal examinations. Hunched posture, piloerection, pale feces and lean appearance was observed at 300 and 600 mg/kg. Maternal findings included body weights, body weight gains, uterus corrected body weights, and food consumption that were statistically significantly reduced in 300 and 600 mg/kg-treated animals. No abnormal findings were noted for dams in the 100 mg/kg group. Statistically significantly lower fetal body weights in both sexes was noted in the 300 and 600 mg/kg groups. An increase of skeletal variations, including 14th full ribs, caudal shift of pelvic girdle and unossified metatarsals was noted in fetuses from animals treated with 300 and 600 mg/kg test article. Variation of the 7th cervical ossification sites was not observed in fetuses from animals treated with 300 and 600 mg/kg. No treatment-related findings were noted in any of the remaining developmental parameters investigated by treatment up to 600 mg/kg. No abnormal developmental findings were noted in fetuses from animals treated with 100 mg/kg. The findings observed in the fetuses from the 300 and 600 mg/kg dose group do not appear to be test-substance specific effects as the changes were only body weight reductions and skeletal variations associated with maternal toxicity. Based on the results of the study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) is 100 mg/kg.