A mixture of: esters of C14-C15 branched alcohols with 3,5-di-t-butyl-4-hydroxyphenyl propionic acid; C15 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate; C13 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Effects on fertility

Description of key information

A fertility and reproduction toxicity study was carried out in rats treated with test article ANOX – BF,  according to OECD test guideline 415, in compliance with GLP.

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 22 1996 to January 17 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Justification for study design:

As per the guideline at the time the study was conducted.

Specific details on test material used for the study:

Not specified

Species:

rat

Strain:

other: Crl CD (SD) BR

Details on species / strain selection:

The Crl CD (SD) BR rat is a rodent of proven experimental validity for the purpose of the study

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
The 120 Crl.CD (SD) BR male and female rats were selected from a group purchased in excess (132 females + 135 males) of the number required for the study from Charles River Italia S.p A, Via Indipendenza, 11, 22050 CaIco (Lecco) On receipt the females were 9 weeks old and weighed 200-225 g; the males were 4-5 weeks old and weighed 100-125 g. The males were delivered on November 8, 1996 with shipping slip no. 07914 and the females on January 3, 1997 with shipping slip no 00046. The animals were housed in the same room where they were mated.
The animals were physically examined on arrival. The body weight was recorded, on arrival, in 20% of the animals and, after about a week in a group comprising 50% of the animals received.
The data relating to weight increase fell within the limits of normal variability for the strain, and thus, taking into account the normal behavior of the animals sampled, the entire group was deemed fit to be used for the experiment.
In our laboratories, the strain used is periodically checked with a known teratogen (cyclophosphamide or hydroxyurea) and is found to be sensitive.

Accommodation
The rats were kept in rooms D 21 and D 19, under the following ambient conditions: temperature 22°C :! 2, relative humidity 60% :! 20, about 10-15 air changes per hour, filtered on REP A 99.99% fiters, artificial lighting with circadian cycle of 12 hours (7 a.m. - 7 pm.) In the event of a main power supply failure an automatic standby power supply was ready to be brought into operation.
The rats were divided by sex and kept in makrolon cages measuring 425x266x150 h mm, type 3D - Tecniplast Gazzada s r.l., Buguggiate - Varese, each fitted with stainless steel cover-feed rack.
Dust-free poplar/fir wood chips, heat processed for resin removal, (type 700/2000 Robina S.n.c , Moncalieri - Torino) were used for bedding.
The producer supplies for each batch of wood chips a certificate of analysis for chemical contaminants.
Contaminants that might interfere with the objective of the study were not expected to be present.
The analytical certificates are filed at REM premises.

Diet and water supply
The animals were fed a diet coded as GLP 4 RF 25 top certificate, produced by Charles River Italia's feed licensee, Mucedola s.d., Settimo Milanese.
The diet was supplemented by the producer with vitamins and trace elements.
According to the analytical certificates provided by the supplier, the contents of the batch/es of the diet used in this study were within :t 5% of the declared values and contaminants were within the limits proposed by the E P A - TSeA (44 FR.44053-44093, July 26, 1979).
The animal feed in compliance with RBM' s SOPs, is analyzed at least twice a year for bacterial contamination.
The diet was available to the animals "ad libitum" Filtered water was distributed by means of an automatic watering valve system The drinking water offered to the animals "ad libitum" came from the municipal water main.
The water is periodically analyzed for microbiological count, heavy metals, other contaminants (e g. solvents, pesticides) and other physical and chemical characteristics.
The acceptance limits of quality of the drinking water were those defined in EEC Directive 80/778.
Contaminants that might interfere with the objective of the study were not expected to be present either in the diet or in the drinking water.
The analytical certificates of animal feed and water are filed at REM premises.

Subdivision of the F0 generation animals into groups and relative dosages
Before treatment was started, all animals were weighed. Animals at the extremes of the weight distribution and animals showing signs of illness were excluded from the study. From the remaining animals, the required number of rats were allocated to the dosage groups by means of a computerized randomization program.
Each animal was identified by an ear tag.
Each cage bore a label indicating experiment number, sex, animal identification number, dosage group color and, during the FO pre-mating period, also the cage number.
During the mating period, the females were examined daily and, as soon as copulation had been ascertained (positive vaginal smear), 8 females of each group were allocated to the Ft and 22 to the Ff subgroup Care was taken to distribute the mated females to the Ft and to the Ff subgroups in progressive order as copulation was ascertained. Each cage housing one pregnant dam also bore the indication of the subgroup, t or f, to which the dam belonged.
The subgroup designed Ft was killed on day 20 of pregnancy and in this group morphologic malformations on the fetuses were studied. The subgroup designed Ff was allowed to litter and killed on day 21 of lactation period with their pups except one male and one female in each litter, tested for the behavioral tests.
The dosages had been established in accordance with the Sponsor and on the basis of previous toxicity studies The highest dosage is the maximum tolerated dose in the 4-week toxicity study in rats. The lowest dosage is the NOEL in the 4-week toxicity study.
The volume of the formulations to be administered to each animal was calculated on the basis of the last body weight recorded
During the lactation period, marks were made on different parts of the pups' bodies with a proper colouring in order to indicate their position within the litter. During the growth period, the rats were identified as their parents besides the marks made on different parts of the body as described before. The group identification color was used on all material (cages, beakers, containers and so on).

Route of administration:

oral: unspecified

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test article preparation
Every day, exact amount of AN OX BF was weighed into suitable graduated polypropylene container and made up to the final volume with vehicle in order to obtain the final concentrations of 5, 25 and 500 mg/ml (groups 2, 3 and 4, respectively). Magnetic stirring was used to obtain homogeneous solution.
The formulates daily prepared were dispensed wrapped in an aluminum foil and administered to animals within 24 h of preparation.

STABILITY
The test article formulates at the concentrations of 5 and 500 mg/ml proved stable for
24 h at room temperature, in the dark (RBM Exp no. 910415).

CONCENTRATION
Test article formulates were tested for concentrations by RBM with an HPLC method.
The methodological specifications were:
HPLC (Merck - Hitachi):
-L-4200 UV/Vis detector
-L -6200 intelligent pump
-AS 2000 autosampler
-D 2500 chromato-integrator
Column: Lichrospher 100 RP-8, 15 cm x 4.6 mm i.d., 5 µm
Column temperature: Room temperature
Mobile phase: acetonitrile/water 98/2 (v/v)
Flow rate: 1 ml/min
Wavelength: 280 nm
Injection volume: 100 µl
Test article formulates were checked for concentration three times/treatment group during the administration period on the formulates prepared on November 27, 1996, on January 21, 1997 and on March 13, 1997 Samples were analysed after extraction in acetonitrile and appropriate dilution in solvent at a final concentration of 0.1 mg/ml.
All analyses were performed in duplicate.

Frequency of treatment:

The test article was administered daily to the FO males from day 70 prior to the mating phase until the end of this phase and to the FO females for 14 days before the start of the mating period and throughout the same. Treatment continued during pregnancy up until day 19 for the females of subgroup Ft killed on day 20 of pregnancy and until the end of lactation (day 21 of lactation) for the mothers of subgroups Ff. The females with positive smear which did not give birth were treated until killing (presumed day 27 of pregnancy). The females without positive smear
were treated up until 27 days after the end of the mating period.

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

30 male and 30 female rats per group.

Control animals:

yes, concurrent vehicle

Details on study design:

MATING
FO - The rats were mated one male with one female, the same male always with the same female, and no others, 7 evenings/week for a maximum of 21 evenings for 16 hours at a time. Day 0 of pregnancy was determined on the basis of the presence of spermatozoa in the vaginal smears taken.

OBSERVATION OF CLINICAL SIGNS AND BEHAVIOR
Clinical signs and behavior were observed and recorded daily, before and after administration, from the start of treatment to the final killing of parent rats of the FO generation. The dams of the Ff subgroup were observed when possible during parturition The birth was observed three times a day during the working time (in the early morning, in the early afternoon and in the late afternoon). If births occurred after the working time (during the night) following day was defined as day 0 of
lactation This criterium was selected because the animals were caged for mating during the night.
The newborn pups of the F1 generation were observed daily from birth up until the sacrifice.
Any deviation from the norm was recorded.

MORT ALITY
The rats found dead were subjected to autopsy to detect the cause of death, whenever possible. Corpora lutea and implantations were counted, whenever possible.
The organs with gross alterations were fixed in formalin for histologic examination, if necessary.

ABORTION
The female rats presenting signs of abortion (vaginal bleeding) were left alive and autopsied at day 20 of pregnancy. Implantations were counted, whenever possible.

BODY WEIGHT
FO - All rats were weighed on a weekly basis during the pre-mating and mating periods.

During pregnancy, the females were weighed on days' 0, 7, 14, 17,20 and during lactation on days 0 (parturition day), 7, 14,21. The body weights of the females found to be pregnant but without positive vaginal smear were excluded from the calculation of the mean body weight values during the pregnancy period.
F1 - All the animals were weighed on days 0, 4, 8, 12 and 21 after birth and on a weekly basis up until the end of behavioral tests.

FOOD CONSUMPTION
During the pre-mating period of the male and female FO generation, food was distributed in weighed amounts. The leftover amounts of the weighed food allocated per cage were recorded once a week in order to calculate food consumption in g/rat/day.
Food consumption was then recorded on days 7, 14, 17 and 20 during the pregnancy period and on days 7 and 14 during the lactation period.

OBSERVATIONS ON THE Ft SUBGROUP
The FO females assigned to the Ft subgroup were killed by cervical dislocation after a preliminary CO2 anesthesia on day 20 of pregnancy.
Observations were made on the following parameters:
- pregnant uterus weight
- number of corpora lutea
- number of resorptions early: only placenta visible late. placenta and embryo visible
- number and sex of viable fetuses
- number and sex of dead fetuses (fetuses without spontaneous movements and
breathing)
- number of implantations
- individual placental weight
- individual fetus weight
A gross external examination was done on all fetuses immediately
Skeletal malformations, anomalies and variants were sought clearing 50% of the fetuses of each litter (1-2)
The remaining 50% of the fetuses of each litter were preserved in Bouin's fluid for examination by Wilson's technique (3).
As far as possible, the distribution per litter for examination by clearing or by Wilson's technique was also by equal sex
Dead fetuses were fixed after the external examination. The observations on fetuses were classified as follows
- malformations: rare and/or normally lethal (such as hydrocephalus, thoracocele, acephalia, amelia, phocomelia, celosomia etc )
- anomalies: more frequent and non-lethal (such as incomplete cranial ossification, hemorrhage, edema, etc.)
- variants: common in the control populations and often definable only in terms of continuously variable gradients (e.g. poor ossification of sternebrae).

OBSERVATIONS ON THE Ff SUBGROUP AND ON THE F1 GENERATION

The FO females assigned to the Ff subgroup were allowed to give birth. On day 0 the pups of each litter were identified by a mark made with an appropriate coloring on different sides of the body; this mark was renewed when necessary. At birth and during lactation, the offspring were observed for:
- external abnormalities at birth
- number of live and stillbirths (ascertained by docimasia)
- mortality ensuing after live birth and ascertained daily. Whenever possible, any pup found dead was examined externally and internally in an attempt to determine the cause of death
- sex determination (day 0)
- pup weight at days 0, 4, 8, 12,21
- appearance of fur (daily from day 4 on)
- eruption of incisors (daily from day 7 on)
- eye opening (daily from day 12 on)
- pinna detachment (daily from day 4 on) the day on which the edge of the pinna became detached
- surface righting reflex (daily from day 4 on)' the pups' ability to return to their normal prone position if placed in a supine position (4)
- pinna reflex (daily from day lIon)' the pinna base was touched with a horsehair; reaction to the stimulus was scored as positive (4)
- corneal reflex (daily from day 12 on): the cornea was touched with a horsehair; eyelid closure in response to the stimulus was scored as positive (4)
- pupillary reflex (daily from day 12 on) after eye opening, a light was slowly beamed toward the eye If the pupil constricted and the rat turned its head to avoid the light, it was considered positive (4)
- traction reflex (daily from day 4 on): a taut, horizontal wire 2 mm in diameter was suspended approximately 25 cm above a flat surface. Each pup was hung on the wire by its front paws. A positive traction response was recorded if the pup was able to grip the wire with at least one back leg within 5 seconds (5)
- grip reflex (daily from day 4 on): if, when hung from a wire, the pup did not fall off within 5 seconds, the grip reflex was recorded as present (5)
- chimney test (day 14) pyrex tubes, 30 cm long, were used. The internal diameter varied with the pup's weight Each tube had a mark 5 cm from its base. A pup was introduced at the base end of the tube laid horizontally near the mark. When the animal had reached the other end of the tube, towards which it was pushed if necessary with a rod, the tube was stood vertically (pup muzzle down). The immediate reaction expected was that the pup should attempt to climb the tube
backwards. The test was positive when the pup reached the mark in less than 30
seconds (6).

On day 21 of lactation the entire F 1 generation was killed except for one male and one female of each litter, selected so as to have a body weight as close as possible to the mean litter weight. If in one litter there was only one pup, this pup was selected; if there were only females or only males, one male or one female were selected. The number of animals in the litter was balanced in the first subsequent litter where this was possible. The pups left alive were weighed weekly and checked daily for clinical signs and mortality. They were observed for descent of testes and for balano-preputial gland separation (daily from day 25 on) and for vaginal opening (daily from day 30 on). At weeks 7-8 from birth they were subjected to the following behavioral tests:

- Inclined plane (week 7 or 8). the apparatus consists of two planes hinged together; one of the planes is used as the base and the other as the inclined plane Along the base there is a series of notches into which a metal stand can be slotted so that the slope of the inclined plane may be varied from 30 to 75 degrees by 5 degree intervals. The rats was placed at the bottom of the inclined plane kept at the lowest inclination. The inclination was gradually steepened and the greatest inclination at which the pup kept its balance for at least 5 seconds was recorded (7).

- Water Y-Maze (week 7 or 8)' the tank, which has arms of equal length and angle of contact, was almost completely filled with water maintained at a temperature of 37° C :l 1 ° C. During the trials, the top was covered with a light-proof lid with the exception of one escape exit which was illuminated (4).
The time taken by each animal to swim through the maze in three successive trials on the same day was measured.
A maximum of 60 seconds was allowed for each trial and any animal that exceeded this time was removed from the water and was considered to have failed the trial and was not considered for the calculation of the mean exit time. Maintained improvement in the swimming time was taken as an indication of learning. Recall ability or memory was assessed by retesting the animals 2 and 7 days after the first trial.
At the end of these behavioral tests the animals underwent necropsy as did the other members of the same litter.

Parental animals: Observations and examinations:

F0 - Males:
They were killed at the end of the mating period. The following organs were removed for possible histological examination: testes, epididymides, seminal vesicles, coagulating gland, prostate (fixed in Bouin's fixative) and pituitar gland (fixed in 10% buffered formalin). The testes were also weighed.

F0 - Females:
Those belonging to the Ft subgroup were killed on day 20 of pregnancy, those belonging to the Ff subgroup after their respective offspring reached 21 days of age The number of corpora lutea and implantations was recorded for the Ft dams, and the number of implantations was recorded for the Ff dams The uterus of apparently non-pregnant females was stained using the Salewski method (8) and examined for the presence of implantation sites.
Ovary, uteri, cervix, vagina and pituitary gland were removed and fixed in 10% formalin solution for histologic examination if necessary The ovaries were also weighed.

Postmortem examinations (parental animals):

The weight of testes was significantly higher in the 1000 mg/kg/day treated males while the percentage value was similar in all experimental groups.
The ovaries weight of the 1000 mg/kg/day treated group was slightly lower than that of the control ovaries, without reaching any significance level.
Testes and epididymides of rats of groups 1 and 4 were histologically examined. No treatment-related modifications were seen in either organ, since their morphological appearance was in all animals within the limits of normality for rats of this strain and similar age used as controls in this or other studies in our laboratories.

Statistics:

See Any other information on materials and methods below for details.

Offspring viability indices:

No important differences were noted in the birth, viability on day 4 and weaning indices and as a consequence in the probability of survival per group between the control and the treated groups.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs or behavioral changes were noted in any experimental group
during the pre-mating, mating, gestation and lactation periods.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (No. 229) of the i 000 mg/kg/day group died owing to difficult parturition (dystocia)
One accidental death was found: female no. 102 of the 10 mg/kg/day group died owing to a wrong gavage

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

PRE-MATING PERIOD:
No differences were found in the body weight gain of treated males and those of the control males during pre-mating, while a slightly not statistically significant lower body weight gain of the females treated with 1000 mg/kg/day and with 50 mg/kg/day was found during the pre-mating
period compared to the control group.

GESTATION PERIOD:
The absolute body weights of the females treated with 50 and with 1000 mglkglday were lower than that of the control group during gestation, to a higher extent in the 1000 mglkglday treated group The significance level was reached during all the pregnancy period for the 1000 mglkg/day group and on days 7 and 14 for the 50 mglkglday group In both cases the final body weights without pregnant uterus were significantly lower than that of the control group.
The body weight gains were always significantly lower in the case of the 1000 mg/kg/day group, while the significance level was reached only from day 0 to day 20 and from day 0 to the final body weight excluding pregnant uterus in the 50 mglkglday.
No statistically significant differences were noted in the absolute body weight of the 10 mglkglday. At this dose a significant lower value was found in the body weight gain from day 0 to day 20 and from day 0 to the final body weight excluding pregnant uterus only in the caesarean sectioned females, where, by chance, there was a body weight of the controls at day 0 higher than that of the control females allowed to litter.
In addition in the controls of the caesarean sectioned females there was also a particularly high number of live fetuses compared to the number of live pups of the controls of the females allowed to litter and to the historical controls. These two findings together were the cause of these incidental statistical significances at 10 mglkg/day.

LACTATION PERIOD:
The body weight of the 1000 mg/kg/day group was still significantly lower at day 0 and at day 7 of lactation, becoming similar to that of the control group thereafter.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

PRE-MATING PERIOD:
The mean daily food consumption of treated males was similar to that of control males, while those of the 1000 mg/kg/day treated females was significantly lower than that of the control females.

GESTATION PERIOD:
The mean food consumption observed in the 50 and in the 1000 mglkg/day group were lower in comparison with the control group, reaching the significance level in the 1000 mg/kglday treated group from day 14 to day 20 of gestation.

LACTATION PERIOD:
No important differences were noted on the mean daily food consumption between control and treated groups.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The length of pregnancy was slightly higher in the 1000 mg/kg/day treated group, where one female also died owing to difficult partrition.
The pregnancy index was lower at 1000 mg/kg/day where 1 female had no live pups at birth
No significant differences were found in the number of implantations in all experimental groups, while the number of live born pups was lower at 1000 mg/kg/day In addition a higher number of still births was present at 1000 mg/kg/day.
As a consequence the mean value per litter of post- implantation losses was higher at 1000 mg/kg/day.
No significant differences were noted in the sex ratio.

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

No dams with late resoiptions or with 100% resoiptions or with dead fetuses were observed in any experimental group. On the other hand a higher number of females with early resorptions was found both in the 50 and in the 1000 mg/kglday treated groups compared to the control group but, while the frequency per group and the mean values per litter of early resoiptions of the 50 mg/glday treated group were similar to those of the control group, in the case of the 1000 mg/kg/day they were higher than controls, reaching the significance level as frequency per group.
In addition both the frequency per group and the mean values per litter of live fetuses were significantly lower at 1000 mg/kg/day compared to controls. As a consequence the mean value per litter of the post-implantation losses was higher at the high dosage compared to control
The percentage of the female fetuses was higher in the 1000 mg/kg/day group than in the control group.
No significant differences were observed in the mean fetal weight and in the mean placental weight. The mean litter weight and the gravid uterus weight at 1000 mg/kg/day were lower than the controls, reaching the significance level as far as the mean litter weight, these findings being related to the lower number of fetuses present in this group.

MALFORMTIONS, ANOMALIES AND VARINTS
One pluri-malformed fetus was observed at the external examination in the 1000 mg/kg/day treated group having ablefaria, acrania, exencephaly, exophthalmia and macrophthalmia. No skeletal malformations were found.
The skeletal anomalies were similar in all experimental groups, while the skeletal variants were higher in the treated groups, but without any dose-relationship. One fetus with malformation (i.e. hydroureter associated with hydronephrosis) was found in the control group and 3 fetuses with hydroureter were found in the 1000 mg/kg/day treated group.
A significant increase was observed in the frequencies per group of visceral variants in the 1000 mg/kg/day group, all related to the urinar tract.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No important differences were noted in the birth, viability on day 4 and weaning indices and as a consequence in the probability of survival per group between the control and the treated groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No important differences were found in the mean body weight of pups belonging to treated and control groups during the lactation and post- lactation periods.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

All animals with the exception of the pups chosen on day 21 of lactation, were killed at the age of 21 days and macroscopically examined for abnormalities.
The pups chosen for behavioral tests were killed, at the end of the behavioral tests
The necropsy was also performed when possible on animals which died during the lactation period.

Histopathological findings:

no effects observed

Description (incidence and severity):

Testes and epididymides of rats of groups 1 and 4, which were fixed in Bouin's fluid, were embedded in paraffin blocks, sectioned at about 5 micrometers thickness, stained with hematoxilin and eosin and microscopically examined.

Other effects:

no effects observed

Description (incidence and severity):

PRE-WEANING MORPHOLOGICAL AND PHYSICAL DEVELOPMENT
AND POST-WEANING SEXUAL DEVELOPMENT:
No differences were noted in fur appearance, pinna detachment, eruption of incisors, eye opening, corneal and pupillary reflexes, and in grip, traction, surface righting and pinna reflex development times between the young of treated dams and those of the control dams.
No differences were observed in the percent of success at the chimney test among
the different experimental groups.
No differences were noted in testes descent, balano-preputial gland cleavage and
vaginal opening among the different experimental groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The time taken to find the exit at the Water Y Maze test was similar in all the experimental groups.
No differences were observed at the inclined plane test among the different experimental groups.

Developmental immunotoxicity:

not specified

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The NOAEL for the F0 parents is 1000 mg/kg/day and for the F1 progeny 1000 mg/kg/day.

Executive summary:

F0 GENERATION

One female treated with 1000 mg/kg/day died owing to diffcult partrition (dystocia). No other compound-related mortality or clinical signs were noted during the study. No effects were noted on the mean body weight and on the mean daily food consumption of treated males during the study while the mean body weight and the mean daily food consumption of the females treated with 50 and 1000 mg/kg/day were lower compared to controls, to a higher extent at 1000 mg/kg/day.

No important differences were noted in the mating and fertility indices among the different experimental groups, and no important effects were noted in the weight of gonads (the weight of testis at 1000 mg/kg/day was higher than controls only as absolute weight and not as percentage of the final body weight at sacrifice). An increase in early resorptions were noted at 1000 mg/kg/day with a decrease of live fetuses. One pluri-malformed fetus was found at 1000 mg/kg/day The length of parturition was slightly longer at 1000 mg/kg/day; in addition diffcult partrition was found in one 1000 mg/kg/day treated female. A higher number of still born with a related lower number of live births was found at 1000 mg/kg/day, while no effects were observed on the postnatal survival and development of F1 live pups The apparent higher frequency of female fetuses was not considered compound related, since such an effect was not found in the females allowed to litter.

CONCLUSION The test article ANOX BF given by oral route to male rats during the pre-mating and mating periods and to female rats during the pre-mating, mating, gestation and lactation periods did not induce any apparent toxic effects on male animals, while the females of the 50 and of the 1000 mg/kg/day had a lower body weight gain and a lower mean daily food consumption. At 1000 mg/kg/day where maternal toxicity was present, an increase of early resorptions and of still birth was also found. No effects on postnatal survival and development of the F1 live pups were noted at any dose. The NOAEL for the F0 parents is 1000 mg/kg/day and for the F1 progeny 1000 mg/kg/day.

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:

K1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

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:

K1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

In this study the effects of ANOX BF were assessed on the reproductive performance of male and female Crl CD (SD) BR rats, when treated in the F0 generation, and on the subsequent morphological, physical and behavioral development of the F1 generation beside to have information on a possible teratogenic activity of the test article.
The doses were 0, 10, 50 and 1000 mg/kg of ANOX BF given by oral route once a day to male and female rats. Dosages were selected in accordance with the Sponsor and on the basis of previous toxicity studies.
Corn oil was administered to the control group with the same schedule.
In the present study, 30 male rats per group were treated from day 70 prior to mating until the end of the mating period and 30 female rats per group were treated for 14 days before the start of the mating period, throughout the same, during gestation up until day 19 for the females of subgroup FO-Ft (females in which information on a possible teratogenic activity could be observed) and up until day 21 of lactation for dams of subgroup FO-Ff (females allowed to give birth and rear their young until weaning). Each male was mated with one female of the same dosage group. During the mating phase, every morning vaginal smear from each female was examined at the microscope and the day in which the presence of spermatozoa was found was considered day 0 of gestation. Eight females with positive vaginal smear were assigned to caesarean section on day 20 of presumed gestation, and twenty-two were allowed to deliver spontaneously. Subsequently, these 22 females were sacrificed after the F1 pups were weaned on day 21 of lactation.

Dams were observed daily for clinical signs, behavior and mortality. Body weight and food consumption were recorded at the scheduled days during the study. All animals were necropsied at death or sacrifice.

On dams killed on day 20 of gestation, the following parameters were recorded:
number of corpora lutea and implantations, number of live fetuses and embryonic/fetal deaths, fetal sex, fetal and placental weight. Fetuses were examined externally for abnormalities. Subsequently about one-half of the live fetuses per litter were sectioned freehand to observe visceral abnormalities (according to the Wilson's technique). The remaining one-half per litter was cleared and stained with alizarin red S for the skeletal evaluation. On rats allowed to litter and rear their young, maternal behavior and delivery were observed. Gestation period and litter data were recorded from birth up to weaning. Pups were observed daily for survival and abnormalities, weighed at the scheduled times during lactation and examined for pre- weaning developmental stages and behavior.
Both parents and young were then killed and macroscopically examined.

F0 GENERATION
One female treated with 1000 mg/kg/day died owing to diffcult partrition (dystocia).
No other compound-related mortality or clinical signs were noted during the study.
No effects were noted on the mean body weight and on the mean daily food
consumption of treated males during the study while the mean body weight and the mean daily food consumption of the females treated with 50 and 1000 mg/kg/day were lower compared to controls, to a higher extent at 1000 mg/kg/day.

No important differences were noted in the mating and fertility indices among the different experimental groups, and no important effects were noted in the weight of gonads (the weight of testis at 1000 mg/kg/day was higher than controls only as absolute weight and not as percentage of the final body weight at sacrifice).
An increase in early resorptions were noted at 1000 mg/kg/day with a decrease of live fetuses.
One pluri-malformed fetus was found at 1000 mg/kg/day.
The length of parturition was slightly longer at 1000 mg/kg/day; in addition diffcult partrition was found in one 1000 mg/kg/day treated female. A higher number of still born with a related lower number of live births was found at 1000 mg/kg/day, while no effects were observed on the postnatal survival and development of F1 live pups
The apparent higher frequency of female fetuses was not considered compoundrelated, since such an effect was not found in the females allowed to litter.

CONCLUSION
The test aricle ANOX BF given by oral route to male rats during the pre-mating and mating periods and to female rats during the pre-mating, mating, gestation and lactation periods did not induce any apparent toxic effects on male animals, while the females of the 50 and of the 1000 mg/kg/day had a lower body weight gain and a lower mean daily food consumption.
At 1000 mg/kg/day where maternal toxicity was present, an increase of early resorptions and of still birth was also found.
No effects on postnatal survival and development of the F1 live pups were noted at any dose.
The NOEL for the F0 parents is 10 mg/kg/day and for the F1 progeny 50 mg/kg/day.

Justification for classification or non-classification

Based on the available information relating to reproductive and developmental toxicity of ANOX BF, the substance does not meet the criteria for classification in accordance with Regulation 1272/2008.