Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 November, 2017 - 22 August, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Physical form / color Pale yellow/solid
Batch number 55503625
Lot number 02/13
Purity 100 %
Storage conditions At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Females nulliparous and non-pregnant: no
- Age at study initiation: Males: 8 to 10 weeks, Females: 10 to 13 weeks
- Weight at study initiation: Males: 235-348 g, Females: 192-231 g
- Fasting period before study: none
- Housing:
Cages with standard, granulated, S8-15 sawdust bedding (J. Rettenmaier & Söhne)
Premating period (5 animals/cage) Makrolon type IV cages
Mating period (one male and one female/cage) Makrolon type III cages
Postmating, gestation and lactation periods (individual) Makrolon type III cages
- Use of restrainers for preventing ingestion (if dermal): n/a
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Eight days between arrival and pre-treatment start. After acclimatization period, the animals were subjected to a 19-day pretest period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30 and 70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/12 hours dark.
IN-LIFE DATES: From: 21 February 2018 To: 22 August 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prepared weekly in Arachis oil and stored at room temperature and in the dark

DIET PREPARATION
- Rate of preparation of diet (frequency): standard Teklad 2014C rat/mouse maintenance diet used
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: not specified

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard vehicle in these studies
- Concentration in vehicle: n/a
- Amount of vehicle (if gavage): 5mL/Kg
- Lot/batch no. (if required): KMO9422, KMO9047
- Purity: not specified
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: vaginal plug and (or) sperm in vaginal smear
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged: Females were separated when evidence of mating was detected
- Any other deviations from standard protocol: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulations prepared at three different concentrations were analyzed twice over the course of the study to verify that they were correctly prepared. Controls were also analysed to confirm the absence of the test item.
The test item was used as analytical standard.
12-mL aliquots (in duplicate) were taken from each formulation to be analysed.
Duration of treatment / exposure:
5-8 weeks
Frequency of treatment:
Once daily
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters: not specified
- Selection of parents from F1 generation when pups were [...] days of age: not specified
- Age at mating of the mated animals in the study: 2 weeks after treatment

Potential indirect exposure to F1 in utero and through the milk during lactation
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control)
Dose / conc.:
1 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of GLP Toxicology Study No. 87-XE-ODBP-10 conducted by the U.S. Army Public Health Command and the preliminary results obtained in the previous non-GLP study PX87XC 7-day Oral (Gavage) Dose-Range Toxicity Study for OECD 421 conducted at Envigo CRS, S.A.U.
- The high dose level of 20 mg/kg was chosen with the aim of inducing some developmental and/or maternal toxicity but not death or severe suffering. This dose level was previously tested in toxicology study No. 87-XE-ODBP-10 and produced signs of systemic toxicity but no mortality or suffering. In study PX87XC this dose level showed no evidence of toxicity or mortality.
- The intermediate and low dose levels were selected as a descending sequence to demonstrate any possible exposure-related response.

- Rationale for animal assignment: Random
Positive control:
none

Examinations

Parental animals: Observations and examinations:
Animals and their cages: Visually inspected twice daily for evidence of reaction to treatment or ill-health.
Blood sampling for hematology or blood chemistry analysis was not performed on animals that died or were sacrificed prematurely.

BODY WEIGHT: Yes
- Time schedule for examinations: Pre-test, Treatment (14 days prior to mating), Mating, Post-mating, Gestation, Lactation

FOOD CONSUMPTION AND COMPOUND INTAKE: weekly

WATER CONSUMPTION AND COMPOUND INTAKE: No
- Time schedule for examinations:

OTHER:
Clinical signs - twice in pre-test, once a week during treatment, mating, and post-mating, day 0, 7, 14 and 20 during gestation, and day 1, 4, 7, and 13 during lactation
Oestrous cyclicity (parental animals):
Dry smears Using inoculation loops during the following phases:
• For 14 days before treatment (all females including spares); animals that fail to exhibit 4-5 day cycles were not be allocated to study.
• Daily from the beginning of treatment period until evidence of mating.
• On the day of necropsy
A cotton swab impregnated in distilled water was used in order to check the evidence of mating during the mating period.
Litter observations:
Clinical observations Observed 24 hours after the considered birth and then daily for evidence of ill-health or reaction to maternal treatment
Litter size Daily from day 1-13 of age
Sex ratio Days 1, 4, 7 and 13 of age
Individual offspring body weights Days 1, 4, 7 and 13 of age
Ano-genital distance Day 1 – all F1 offspring
Nipple/areolae count Day 13 of age – male offspring
Postmortem examinations (parental animals):
F0 Males After final investigations completed (after 5 weeks of treatment)
F0 Females failing to produce viable litter (not pregnant females) Day 25-26 after mating
Females killed at termination Day 14-16 of lactation
Postmortem examinations (offspring):
Selected offspring for thyroid hormone analysis – Day 4 of age (two females per litter where possible)
Scheduled sacrifice - Day 13-15 of age
Statistics:
Bartlett's test, Williams' test, Dunnetts test.
Linear-by-linear test (Cytel, 1995).
Cochran-Armitage test (Cytel, 1995).
Reproductive indices:
Estrous cycle, pre-coital interval, mating performance, fertility and gestation length.
Offspring viability indices:
Survival indexes, litter size, sex ratio, body weight, clinical signs, ano-genital distances or external examination.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The death of two females (numbers 75 and 66) could be considered occasional and not treatment related given that no mortality was recorded at the high dose, due to the incidence observed in the study and to the fact that no test item related effects were observed in these animals during the study. The major factor contributing to their death remained undetermined histologically.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
T4 and TSH determinations: a decrease was recorded when compared to Control in adult males (at all doses but mainly at 20 mg/kg/day) and females (at 20 mg/kg/day). It was also observed at 20 mg/kg/day in offspring males and females on Day 13 of lactation. Despite the effect observed at 20 mg/kg/day in T4 and TSH determinations, thyroid and parathyroid glands weights were unaffected by treatment. Based on the fact that what was recorded was a decrease in the levels of TSH analyzed and not an increase (which would be related to a possible effect attributable to the components that alter the hormonal function at the level of generation of hypertrophy / follicular hyperplasia) and that no macroscopic findings have been recorded in the pituitary or thyroid / parathyroid glands, this finding cannot be considered as a relevant adverse effect.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
During treatment one female at 1 mg/kg/day (number 62) and one at 5 mg/kg/day (number 76) showed irregular cycles and one female administered at 5 mg/kg/day (number 70) was acyclic.
At termination, all reproductive phase females (pregnant) showed diestrus with the exception of 1/9 (number 68) at 1 mg/kg/day and 2/9 (numbers 70 and 71) at 5 mg/kg/day. These females showed different stages of estrous cycle, which indicated they had recovered the cycle.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

There was no effect of treatment on the pre-coital interval: all animals mated within four days of mating at the first estrous, with the exception of one female at 5 mg/kg/day, whose pre-coital interval extended to 14 days of mating with one male and 2 days of mating with a second male.
Mating performance was 100% for all groups except 5 mg/kg/day males, for which it was 90%.
Fertility was 100% at 0 and 20 mg/kg/day and 90% (9/10) at 1 and 5 mg/kg/day for males and females. These differences cannot be taken into account based on occurrence and lack of dose response.
Gestation length was within the normal range in all treated groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No effects seen in any of the parameters tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
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:
no effects observed
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

There was no effect on offspring growth. There were no offspring clinical or necropsy signs observed which were indicative of a reaction to DNAN, and there was no relevant effect on litter size, sex ratio, survival indices, body weights, ano-genital distance or nipple areolae.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No effects seen in any of the parameters tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, the effects of oral (gavage) administration of DNAN to Wistar rats receiving 1, 5 or 20 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:
Repeated dose toxicity:
- The No Observed Adverse Effect Level (NOAEL) for repeated dose toxicity was considered to be 20 mg/kg/day for males and females, based on the absence of histopathological findings but the non-considered adverse effects in the T4 and TSH determinations.
Reproductive / developmental toxicity:
- The No Observed Adverse Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 20 mg/kg/day, taking into account that there was no effect on estrous cycle, pre-coital interval, mating performance, fertility and gestation length or in the offspring on survival indexes, litter size, sex ratio, body weight, clinical signs, ano-genital distances or external examination.