Potassium titanium oxide (K1.88-2.13Ti8O16.94-17.07)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March to 30 December 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.35 (Two-Generation Reproduction Toxicity Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Rat: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P) 5-6 wks
- Weight at study initiation: (P) Males: 157-186 g.; Females: 123-142 g.
- Fasting period before study: no
- Housing:
Pre-mating Animals were housed in groups of 4 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 4 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation Offspring was kept with the dam until termination of the dams in Macrolon cages (MIII type, height 18 cm)..
General Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (e.g. ad libitum): Free access to prepared diets. During the acclimatization period, animals had free access to the same diet (without the test substance) received from the supplier in pelleted form (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.
- Water (e.g. ad libitum): Free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 5 days prior to start of treatment.
- Health check F0: A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.3 - 22.7°C
- Humidity (%): 21 - 94%.
Temporary deviations from the minimum temperature, and minimum and maximum level of relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day.

IN-LIFE DATES: From: 19 March to 30 December 2009

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance.

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared at least once every 8 days.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were kept at room temperature in the diet store room in the animal house.

Details on mating procedure:

Following a minimum of 70 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates for the F0-generation. For the F1-generation parentage was known through mating records maintained in the raw data). Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 15 days was allowed for mating. If no evidence of copulation was obtained after 10 days, the female was placed with another male (for an additional five days) of the same treatment group who had successfully mated, again avoiding sibling mating.

- M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 15 days.
- Proof of pregnancy: intravaginal copulatory plug or sperm in vaginal smear; referred to as day 0 post-coitum.
- After 10 days of unsuccessful pairing replacement of first male by another male of the same treatment group with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon cages (MIII type, height 18 cm).
No vaginal lavage was performed on the following occasions: Day 3 of mating: one F0-female of Group 1, Day of necropsy: four F1-females of Group 1, two F1-females of Group 3. There was sufficient information available for evaluation.

Since no treatment-related fertility or reproduction effects were found, additional mating was not performed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted in weeks 2, 11, 22, 33*, 35 and 36, according to a validated method. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).

* Analysis of the diet used for Group 4 in Week 33 of the study revealed an accuracy which was below the target concentration of 80% (i.e. 77%) and a coefficient of variation which was higher than 10% (i.e. 14%). To investigate this further, the following additional measurements were performed:
For preparation of the diet, a different container of the test substance (container A9-2) was used than for the preparation of the procedural recovery samples during diet analyses in Week 33 (container A7). The test substance in both containers was of the same batch. However, the test substance in container A7 was delivered before the start of the study, whereas container A9-2 was delivered at NOTOX on 02 October 2009. To investigate whether there was a difference between the test substances in the two containers, procedural recovery samples prepared from the two containers were measured on 30 November 2009 (Week 33). Comparison showed no difference. Subsequently, analysis (acc + hom) of the Group 4 diet preparation of Week 33 was repeated in Week 35. For this approximately 10 gram of the frozen back-up random sample was used.
In addition, diets prepared for Groups 1-4 in Week 35 were analysed. Analysis of the Group 2 diet revealed a coefficient of variation which was higher than 10% (i.e. 17%). To investigate this further, analysis of diets prepared for Groups 1-4 in Week 36 was performed. Furthermore, Group 1 extracts were spiked with analytical standard to find out whether the relatively high recovery for the procedural recovery samples was due to matrix of the samples.

PROCEDURAL RECOVERY SAMPLES
Mean recoveries of the procedural recovery samples were between 101% and 127% as a result of the difference in matrix between calibration solutions and pretreated test samples. It was therefore decided to correct the results of the test samples for the mean recovery of the procedural recovery samples measured on the same day as the test samples.

Comparison of procedural recovery samples prepared from different containers (A7 and A9-2) of the same batch of the test substance showed no difference between the two containers.

TEST SAMPLES
Accuracy of preparation:
A small response was measured in the Group 1 diets. This response was a background response from the blank diet.

The concentrations analysed in the diets of Groups 2, 3 and 4 in Weeks 2, 11, 22, 33, 35 and 36 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%) except for the diet of Group 4 prepared for use in Week 33. The mean accuracy for this diet was outside the 80-120% range (i.e. 77% of target). The value of < 80% was accepted since reanalysis of the back-up sample revealed a mean accuracy of 88%. The result demonstrated that the Group 4 diet was prepared correctly.

Homogeneity:
The diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ? 10%) in Weeks 2, 11, 22, 33, 35 and 36, except for the Group 4 diet prepared for use in Week 33 and the Group 2 diet prepared for use in Week 35. These diets had a coefficient of variation of 14 and 17%, respectively. However, the impact of the slight inhomogeneities was considered to be negligible, since they were relatively small and occurred after completion of prenatal development. Moreover, no treatment-related effects on pre/post-natal development were noted in the first generation.

RESIDUE SAMPLES
In the residue samples, a small response was observed which was comparable to the response in the Group 1 diets and to the response in blank powder diets which had not been in contact with the pelletizing machine. Therefore, it can be concluded that the cleaning procedures applied after diet preparation were sufficient.

Duration of treatment / exposure:

The F0-generation was exposed for a minimum of 70 days prior to mating and continuing until euthanasia. The F1-generation was potentially exposed to the test substance in utero, through nursing during lactation and directly following weaning. After weaning, pups were treated for a minimum of 70 days prior to mating and continuing until euthanasia. The F2-generation was potentially exposed to the test substance in utero and through nursing during lactation.

Frequency of treatment:

Ad libitum

Details on study schedule:

- F1 parental animals not mated until 70 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 15-16 weeks (F0) and 13 weeks (F1)

Dose / conc.:

0 ppm

Dose / conc.:

1 500 ppm

Dose / conc.:

5 000 ppm

Dose / conc.:

15 000 ppm

No. of animals per sex per dose:

24

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected based on results of the dose range finding study (NOTOX Project 490623).
- Rationale for animal assignment: random

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
At least once daily, detailed clinical observations were made in all animals. The time of onset, degree and duration was recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1, 4, 7, 14 and 21.
No body weight was determined for one F0-female of Group 2 on Days 22 and 36 of the mating period. There was sufficient information available for evaluation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Weekly, for males and females. During Week 1 and Week 8 (pre-mating) of the study, food consumption was determined twice. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1, 4, 7, 14 and 21.
The amount of test substance incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period. The actual test substance intake for each period was estimated based on the body weight and food consumption values.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

For two females (F1, Group 3) no food was left on the morning of necropsy. Therefore, they had no free access to food during the last night of lactation. As food was present during the last animal check the day before, it was concluded that this deviation was slight and of a short duration. The study integrity was not adversely affected. These two animals were exposed to the test substance during a slightly shorter period on this last day of treatment.

WATER CONSUMPTION: No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect on water intake was suspected.

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 21 days prior to initiation of the mating period and until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also prepared to determine the stage of estrous.

No vaginal lavage was performed on the following occasions: Day 3 of mating: one F0-female of Group 1 and on Day of necropsy: four F1-females of Group 1 and two F1-females of Group 3. There was sufficient information available for evaluation.

The stages include e (=estrus), d (=di-estrus), m (=met-estrus), p (=pro-estrus), u (= unable to determine estrous stage/length of estrous cycle) and s (= smeared positive for sperm).

An analysis of the female cycle pattern was reported. The cycle patterns were classified as:
R = Regular (all cycles either 4 or 5 days);
IR = Irregular (at least one cycle of 2, 3 or 6-10 days, irrespective of the number of 4-5 day cycles);
A = Acyclic (least 10 days without estrus, beginning prior to pairing irrespective of the number of 4-5 day cycles);
EE = Extended Estrus (at least 4 consecutive days of estrus);
ED = Extended Di-Estrus during pairing (at least 5 consecutive days of di-estrus during pairing).

Cycle classifications for individual animals were based on the length and stage of each estrous cycle (beginning with the first day of dose administration until evidence of mating was detected).

Sperm parameters (parental animals):

Parameters examined in [F0- and F1] male parental generations:
For all surviving males, the following assessments were performed:
From all males, sperm samples were taken from the proximal part of the vas deferens (right):
1. Sperm motility was assessed from all samples.
2. Sperm smears for morphological evaluation were fixed of all samples. Abnormal forms of sperm from a differential count of 200 spermatozoa (if possible) per animal were recorded. Evaluation was performed for all samples of the control and high dose group. In the absence of a treatment-related effect, the intermediate dose groups were not assessed.
One testis and one epididymis (left) from all males were removed, placed in labelled bags, and kept in the freezer at ?-15°C. After thawing the left testis and epididymis were weighed, homogenized and evaluated for sperm numbers.
In the case of any abnormalities in the testes and/or epididymidis (left), the left side organ(s) were fixed in modified Davidson's, and the right side organ(s) were used for evaluation of sperm numbers.
If abnormalities were found in testes and/or epididymides (both sides), both these organs were fixed in modified Davidson's solution. No evaluation of sperm numbers was performed.
Evaluation was performed for all samples of the control and high dose group. In the absence of a treatment-related effect, the intermediate dose groups were not assessed.

In summary:
Testis weight, epididymis weight, sperm concentration in testes, sperm concentration in epididymides, sperm motility and sperm morphology.

Litter observations:

STANDARDISATION OF LITTERS
To reduce variability among the litters, on Day 4 of lactation, eight pups from each litter of equal sex distribution (if possible) were randomly selected. For litters consisting of fewer than eight pups, adjustment for litter size was not performed.

PARAMETERS EXAMINED
The following parameters were examined in [F1 and F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities:
- Mortality / Viability: The numbers of live and dead pups at the First Litter Check (= check at Day 1 of lactation) and daily thereafter were determined. Animals showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights Live pups were weighed during lactation on Days 1, 4, 7 and weekly thereafter.
- Sex: Was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).
- Balanopreputial separation: Each selected male pup (24 rats/group) was observed for balanopreputial separation (prepuce opening) beginning on postnatal Day 35 (Korenbrot, 1977). Examination of the males continued daily until balanopreputial separation was present. The body weight of each male was recorded on the day of acquisition of balanopreputial separation.
- Vaginal perforation: Each selected female pup (24 rats/group) was observed for vaginal perforation (vaginal opening) beginning on postnatal Day 25 (Adams, 1985). Examination of the females continued daily until vaginal perforation was present. The body weight of each female was recorded on the day of acquisition of vaginal perforation.
- Anogenital distance: In the absence of treatment related effects in the sex ratio or sexual maturation of the F1-generation, no measurement of the anogenital distance was performed in the F2-pups.

From one pup (F0, Group 3), no clinical signs or body weights were available from Days 1-3 of lactation. Based on the absence of clinical signs and a normal body weight recorded on Day 4, it was concluded that this pup developed well until Day 3. For evaluation, sufficient data was available from its siblings.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
F0-and F1-males were killed as soon as possible after delivery of the litters.
F0-and F1-females were killed on Day 21 of lactation or shortly thereafter.
Since no treatment-related fertility or reproduction effects were found, additional mating was not performed.

Females showing no evidence of copulation were killed approximately 28 days after the last day of the mating period.
Non-pregnant females were killed on Day 25-27 post-coitum.

In case a female was not pregnant, the uterus was stained using the Salewski technique (Salewski, 1964) in order to determine any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

At the time of necropsy, a vaginal lavage was taken to determine the stage of estrus.

One F0-female of Group 2 and one F1-female of Group 3, showing no evidence of copulation, were killed 27 and 30 days, respectively, after the last day of the mating period, instead of approximately 21 days after the last day of the mating period. Longer treatment was considered not to have adversely affected the macroscopic or microscopic findings.

GROSS NECROPSY
Gross necropsy consisted of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

At necropsy, no terminal body weights were determined for two animals. One F1-female of Group 2 was sacrificed on lactation Day 21. Therefore, the body weight as determined on lactation Day 21 could be used as terminal body weight. As a consequence of the missing terminal body weight for the F1 female of Group 4, no organ to body weight ratios could be calculated. However, sufficient data from other Group 4 animals was available for a proper evaluation.

Of all paired females, the number of former implantation sites in the uterus was assessed.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively:
Identification marks: not processed
(Pituitary gland)
Adrenal glands
Prostate gland
(Brain (cerebellum, mid-brain, cortex))
Seminal vesicles
Cervix
(Spleen)
Coagulation gland
Testes (1)*
Epididymides (1)*
(Thyroid including parathyroid (if detectable))
(Kidneys)
Uterus
(Liver)
Vagina
Ovaries
All gross lesions

* Testes and epididymis (right only) were fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial)(all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
If there were gross findings in the testes and/or epididymidis (left), the abnormal organ(s) were fixed in modified Davidson's solution as described above, and testes and/or epididymides (right) were used for evaluation of sperm parameters (for details see section 6.7.3.).
If abnormalities were found in testes and/or epididymides (both sides), both these organs were fixed in modified Davidson's solution as described above.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity or target organ involvement were noted at macroscopic examination.

A few tissues/organs were not available for histopathology and organ weights. These tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Tissues are listed in the raw data and pathology report. Sufficient tissues were available for evaluation.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age or shortly thereafter.
Pups, younger than 7 days, were killed by decapitation. All remaining pups were sacrificed using an oxygen/carbon dioxide procedure.
Culling was performed on Day 4 of lactation or shortly thereafter. The remaining pups (excluding F1-pups selected for mating) were killed at Day 21 post partum or shortly thereafter.
- Animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Stillborn pups and pups dying between birth and Day 4 of lactation were sexed and dissected (including the heart and the brain examined by a mid-coronal slice) by a technique described by Stuckhardt and Poppe (Stuckhardt, 1984). These examinations were only performed when practically possible (e.g. in the absence of cannibalism, autolysis). Culled offspring was sexed and externally examined with emphasis on developmental morphology. The stomach was examined for the presence of milk. All nonselected F1 and F2 weanlings were sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs with emphasis on developmental morphology. Descriptions of all macroscopic abnormalities were recorded. All gross lesions were collected and placed in 10% buffered formalin.

GROSS NECROPSY
-Gross necropsy consisted of thoracic and abdominal tissues and organs with emphasis on developmental morphology.

HISTOPATHOLOGY / ORGAN WEIGTHS
The following organ weights (and terminal body weights) were recorded from one pup/sex/litter (the first male and female pup of each litter, if possible) from both generations on the scheduled day of necropsy: brain, spleen and thymus. After weighing, samples of these organs were fixed in 10% buffered formalin for possible future analysis.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.
- The T-test (Gossett, 1908) was applied for sperm concentrations in the testis and epididymis.
- For the F1-generation, sperm concentration in the testis, absolute brain organ weight (males), and number of implantation sites were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal, 1952) to determine intergroup difference. Since the results of the ANOVA were significant (p<0.05), the Wilcoxon test (Wilcoxon, 1945) was applied to the data to compare the treated group(s) and control group.
- The percentage of motile spermatozoa, progressive motile spermatozoa and sperm with normal morphology were subjected to the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate for the comparison of the treated groups and control group (both F0- and F1-generation).

All tests were two-sided and in all cases p<0.05 was accepted as the level of statistical significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might have been rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

Percentage mating males: Number of males mated/Number of males paired x 100
Percentage mating females: Number of females mated/Number of females paired x 100
Fertility index: Number of pregnant females/Number of females paired x 100
Conception rate: Number of pregnant females/Number of females mated x 100
Gestation index: Number of females bearing live pups/Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Offspring viability indices:

Percentage of postnatal loss Days 0-4 post partum: Number of dead pups on Day 4 post partum/Number of live pups at First Litter Check x 100
Percentage of breeding loss Day 5 until weaning: Number of dead pups between Days 5 and 21 post partum/Number of live pups on Day 4 post partum x 100
Percentage live males at weaning: Number of live male pups on Day 21 post partum/Number of live pups on Day 21 post partum x 100
Percentage live females at weaning: Number of live female pups on Day 21 post partum/Number of live pups on Day 21 post partum x 100
Viability index: Number of live pups on Day 4 post partum/Number of pups born alive x 100
Weaning index: Number of live pups on Day 21 post partum/Number of live pups on Day 4 post partum x 100

Clinical signs:

no effects observed

Description (incidence and severity):

Incidental findings included alopecia, scabbing and/or scaling of various body parts. These findings are not uncommon in rats of this age and strain used in this type of study, and because
they were limited to only 1-2 animals per sex from each treatment group, they were considered not to be toxicologically relevant.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically significant changes in body weights and body weight gain were noted up to 15000 ppm. The slightly higher body weights and/or body weight gains recorded in females of Groups 3 and
4 on Days 15, 22 and/or 36 of the pre-mating period were considered not to be toxicological relevant as values remained within the normal range. Moreover, a decrease rather than an
increase in body weight would have been expected in case of toxicity. At 15000 ppm mean body weight gain was significantly lower than controls on Day 14 of the post-coitum period. Since this decrease was only slight and transient, no biological significance was attached to this finding.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no treatment related effects on estrous cycle duration or classification. The percentage of females per group that were classified as having a ‘regular’ cycle was 87.5, 95.8, 79.2 and 87.5 % at 0, 1500, 5000 and 15000 ppm, respectively. In these animals, the incidence of irregular cycle was 8.3, 4.2, 20.8 and 12.5%, respectively. In the absence of a dose-effect relationship, the lower percentage of females with a regular cycle in Group 3 was not considered toxicologically relevant. Furthermore, one control female showed an acyclic

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed on spermatogenesis endpoints in F0-males. These spermatogenesis endpoints included mean testicular and epididymal sperm concentrations, morphology (Group 1 and 4) and motility and progressive motility (all groups).

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproduction parameters were unaffected by treatment up to 15000 ppm TERRACESS P. Mating, fertility, precoital time, conception rate, and number of implantation sites were comparable between treated and control animals.

OBSERVATIONS F0-GENERATION
F0-GENERATION Mortality:
No mortality occurred.

F0-GENERATION Clinical signs:
No toxicologically relevant clinical signs were noted up to 15000 ppm.

Incidental findings included alopecia, scabbing and/or scaling of various body parts. These findings are not uncommon in rats of this age and strain used in this type of study, and because they were limited to only 1-2 animals per sex from each treatment group, they were considered not to be toxicologically relevant.

F0-GENERATION Body weights:
No toxicologically significant changes in body weights and body weight gain were noted up to 15000 ppm.

The slightly higher body weights and/or body weight gains recorded in females of Groups 3 and 4 on Days 15, 22 and/or 36 of the pre-mating period were considered not to be toxicological relevant as values remained within the normal range. Moreover, a decrease rather than an increase in body weight would have been expected in case of toxicity.

At 15000 ppm mean body weight gain was significantly lower than controls on Day 14 of the post-coitum period. Since this decrease was only slight and transient, no biological significance was attached to this finding.

F0-GENERATION Food consumption:
No toxicologically significant changes in food intake (absolute and relative to body weight) were noted up to 15000 ppm.

The slightly higher food intake (before and after allowance for body weight) noted in animals of Groups 2-4 (both sexes) on several occasions was not considered toxicologically relevant as values remained within the normal range. Moreover, if toxicity would be present, a reduction in food consumption would be expected instead.

F0-GENERATION Test article intake:
The mean test article intake values (mg substance/kg body weight/day) are summarized in attached table:
Mean test article intake when corrected for nominal dose level: see attachment; Table 1.

F0-GENERATION Macroscopic examinations:
No macroscopic findings were considered attributable to treatment with TERRACESS P.

Necropsy findings that were noted among control and/or treated animals included testes and epididymides enlarged, reduced in size, and/or flaccid, yellowish or greenish foci or nodules on the epididymides (soft) or epididymal adipose tissue (hard), enlarged liver, tan foci on the lateral liver lobes, diaphragmatic hernia of the liver papillary process, dark red foci on the pancreas, dark red or black-brown discolouration of the adrenal glands, thickened ureter, dark red discolouration of the right uterine horn, and alopecia of various body parts. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological significance.

Uterus containing fluid was noted for 3, 2, 2 and 3 females in Groups 1, 2, 3 and 4, respectively. This is a common finding related to a stage in the estrous cycle and does not reflect any sign of toxicity.

F0-GENERATION Organ weights:
There were no changes in organ weights that were considered to be of toxicological significance.

The following statistically significant changes between mean organ weights of treated and control animals were noted: increased testes weight in Group 3 males (absolute and relative), and increased kidney weight (absolute) and decreased ovary weight (absolute and relative) in Group 4 females. These findings were considered not to be a sign of toxicity, since there was no treatment-related distribution (testes) and/or all organ weights remained within the normal range of biological variation seen for animals of the same age and strain used in this type of study. Moreover, microscopic examination of the ovaries and testes did not reveal any treatment-related changes.

F0-GENERATION Microscopic examination:
No treatment-related microscopic findings indicative of toxicity were identified in the reproductive organs of the F0-generation. All microscopic findings recorded were considered to be within the range of background pathology encountered in Wistar Han rats of these ages.

The reproductive organs were examined from all animals that failed to mate, conceive, sire or deliver healthy offspring (17 males and 8 females).These animals were spread across the dose groups with no relationship to treatment. Findings in F0-animals suspected as infertile were regarded as incidental and not responsible for the suspected infertility.

F0-GENERATION Sperm motility, concentration and morphology:
No treatment-related effects were observed on spermatogenesis endpoints in F0-males. These spermatogenesis endpoints included mean testicular and epididymal sperm concentrations, morphology (Group 1 and 4) and motility and progressive motility (all groups).

F0-GENERATION Estrous cycle:
There were no treatment related effects on estrous cycle duration or classification.

The percentage of females per group that were classified as having a ‘regular’ cycle was 87.5, 95.8, 79.2 and 87.5 % at 0, 1500, 5000 and 15000 ppm, respectively. In these animals, the incidence of irregular cycle was 8.3, 4.2, 20.8 and 12.5%, respectively. In the absence of a dose-effect relationship, the lower percentage of females with a regular cycle in Group 3 was not considered toxicologically relevant. Furthermore, one control female showed an acyclic cycle.

F0-GENERATION Reproduction:
Reproduction parameters were unaffected by treatment up to 15000 ppm TERRACESS P.

Mating, fertility, precoital time, conception rate, and number of implantation sites were comparable between treated and control animals.

For reproduction Data F0-generation: see attachment; Table 2.
For fertility F0-generation: see attachment; Table 3.

F0-GENERATION Breeding data:
There were no toxicologically-relevant effects on gestation index and duration.

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

No treatment-related effects on postnatal pup development (dead and living pups at first litter check, sex ratio, postnatal loss (until lactation Day 4), breeding loss (lactation Day 5-21), viability index and weaning index, clinical signs, body weight, age and weight upon reaching vaginal patency or balanopreputial separation, macroscopy and organ weights) were noted.


OBSERVATIONS F1-GENERATION (PARENTAL ANIMALS)
F1-GENERATION Mortality:
There were no treatment-related deaths during the study period.

One female in Group 2 was sent to necropsy on Day 4 of lactation because the only surviving pup of this dam was killed in extremis due to a poor condition.

F1-GENERATION Clinical signs:
There were no treatment-related clinical signs noted up to 15000 ppm.

For one female in Group 2 piloerection and a pale appearance was noted on lactation Days 1 and 2. In the period before and during delivery no clinical symptoms had been noted for this dam. In addition, body weight, body weight gain and food intake were within the normal range during the post-coitum period. At first litter check eleven out of twelve pups were found dead. The only surviving pup was in a poor condition (i.e. cold appearance, (very) small size, no milk in the stomach, relatively low body weight on lactation Days 1 and 4, no body weight gain). Based on these data it is likely that the dam was suffering from complications shortly after delivery. Since it was an isolated case without any dose-response relationship, it was considered to be a fortuitous finding with no toxicological relevance.

A few animals in the treated groups showed alopecia. In addition, for one male in Group 2 chromodacryorrhoea of the right eye was noted. At the low incidence and/or in the absence of a dose-response relationship these findings were considered not to be toxicologically significant.

F1-GENERATION Body weights:
No toxicologically relevant effects on body weights were noted up to 15000 ppm.

At start of the pre-mating phase, Group 2-4 animals (both sexes) showed lower mean body weights as compared to animals of the control Group 1 (only statistically significant for Group 4 females). This trend continued, reaching statistical significance in males at 15000 ppm (Group 4) on pre-mating Days 8-29. In addition, a trend towards higher body weight gains was recorded for all treated groups (both sexes) as compared to control animals during pre-mating (only statistically significant for Group 4 females). These changes in body weight and body weight gain were caused by the fact that a few pups selected for Groups 2-4 were 8-11 days younger than the youngest pups selected for Group 1. Due to their younger age, these pups had a lower initial body weight and showed a higher body weight gain as compared to the older ones during the first weeks of pre-mating. At the end of the pre-mating period mean body weights were normalized in all treated groups.

The higher body weight gain noted in females at 1500 ppm (Group 2) on post-coitum Day 20 was considered not to be a treatment-related effect. The statistical significance was most likely caused by the fact that the mean body weight gain recorded for control animals was relatively low.

The higher body weight gain noted in males at 5000 ppm (Group 3) on mating Day 15 was considered to be a fortuitous finding since the change as compared to controls was very slight and no dose-effect relationship could be established.

F1-GENERATION Food consumption:
No toxicologically significant changes in food intake (absolute and relative to body weight) were noted up to 15000 ppm.

The slightly higher mean food intake (before and after allowance for body weight) noted in animals of Groups 2-4 (both sexes) on several occasions was not considered toxicologically relevant as values remained within the normal range. Moreover, if toxicity would be present, a reduction in food consumption would be expected instead.

At the individual level, absolute food consumption was relatively low for one cage of males in Group 4, especially in the first week of pre-mating. This lower mean food intake was most likely due to the fact that two out of four cage mates had a relatively low initial body weight and thus consumed less food.

F1-GENERATION Test article intake:
The mean test article intake values (mg substance/kg body weight/day) are summarized in attached table:
Mean test article intake when corrected for nominal dose level; see attachment: Table 4.

F1-GENERATION Macroscopic findings:
There were no macroscopic findings that were considered attributable to treatment with TERRACESS P.

Testes, epididymides and/or seminal vesicles reduced in size and/or gelatinous were noted for three and two males in Groups 2 and 4, respectively. Furthermore, for one male in Group 2 a yellowish, hard nodule on the epididymal adipose tissue was noted. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.

For one male in Group 2, missing left seminal vesicle was recorded. At this low occurrence and in the absence of a dose-effect relationship, it was considered a fortuitous finding.

Incidental findings included liver enlarged in size or with accentuated lobular pattern, liver papillary process enlarged in size and with dark-red discolouration, watery-clear cyst on the ovaries, dark-red discolouration of the mesenteric lymph node, and isolated reddish foci on the thymus. Due to the isolated nature of these findings, and in the absence of a dose-response relationship, they were not considered to be treatment-related.

Alopecia and/or scabbing of various body parts were noted for 1-2 females in Groups 1, 2 and 4 each. As it occurred only incidentally and due to the fact that control animals as well as treated animals were affected, it was considered a fortuitous finding. Moreover, alopecia and scabbing is more often seen for rats of this age and strain housed under comparable conditions.

Uterus containing fluid was noted for three females each in Groups 2-4. This is a common finding related to a stage in the estrous cycle and does not reflect any sign of toxicity.

F1-GENERATION Organ weights:
There were no changes in organ weights that were considered to be of toxicological significance.

The following statistically significant changes between mean organ weights of treated and control animals were noted: increased adrenal weights in males of Group 2 (relative) and Group 3 (absolute and relative) and females of Group 2 (absolute), and increased liver weights in females of Group 2 (absolute and relative) and Group 4 (relative). These findings were considered not to be a sign of toxicity, since no dose-response relationship could be established and/or all organ weights remained within the normal range of biological variation seen for animals of the same age and strain used in this type of study.

F1-GENERATION Microscopic examination:
No treatment-related microscopic findings indicative of toxicity were identified in the reproductive organs of the F1-generation. All microscopic findings recorded were considered to be within the range of background pathology encountered in Wistar Han rats of these ages.

There was no adverse effect of treatment with the test item on the number of primordial follicles in the ovaries of the sampled females.

The reproductive organs were examined from all animals that failed to mate, conceive, sire or deliver healthy offspring (16 males and 9 females). These were spread across the dose groups with no relationship to treatment. In male no. 231 (Group 2) bilateral seminiferous atrophy (marked/massive) of the testes was recorded, which was the cause of infertility for this animal. Findings in the remaining animals were regarded as incidental and not responsible for the suspected infertility.

F1-GENERATION Sperm motility, concentration and morphology:
No treatment-related effects were observed on spermatogenesis endpoints in F1-males. These spermatogenesis endpoints included mean testicular and epididymal sperm concentrations, morphology (Groups 1 and 4), motility and progressive motility (all groups).

The statistically significant changes noted in high dose males (Group 4) for epididymal sperm concentration, left testis weight and sperm morphology were not considered to be a sign of toxicity, as changes were small, remained within the normal range of biological variation seen for animals of the same age and strain used in this type of study and/or represented an improvement rather than deterioration.

F1-GENERATION Estrous cycle:
There were no treatment related effects on estrous cycle duration or classification.

The percentage of females per group that were classified as having a ‘regular’ cycle was 87.5, 95.8, 95.8 and 87.5 % at 0, 1500, 5000 and 15000 ppm, respectively. In these animals, the incidence of irregular cycle was 12.5, 4.2, 4.2 and 12.5%, respectively.

F1-GENERATION Reproduction:
Reproduction parameters were unaffected by treatment up to 15000 ppm TERRACESS P.

Mating, fertility, precoital time, conception rate, and number of implantation sites were comparable between treated and control animals.

For reproduction Data F1-generation: see attachment; Table 5.
For fertility F1-generation: see attachment; Table 6.

F1-GENERATION Breeding data:
There were no toxicologically-relevant effects on gestation index and duration.

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 15 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs noted up to 15000 ppm. For one female (no. 341) in Group 2 piloerection and a pale appearance was noted on lactation Days 1 and 2. In the period before and during delivery no clinical symptoms had been noted for this dam. In addition, body weight, body weight gain and food intake were within the normal range during the post-coitum period. At first litter check eleven out of twelve pups were found
dead. The only surviving pup was in a poor condition (i.e. cold appearance, (very) small size, no milk in the stomach, relatively low body weight on lactation Days 1 and 4, no body weight gain). Based on these data it is likely that the dam was suffering from complications shortly after delivery. Since it was an isolated case without any dose-response relationship, it was considered to be a fortuitous finding with no toxicological relevance.
A few animals in the treated groups showed alopecia. In addition, for one male in Group 2 chromodacryorrhoea of the right eye was noted. At the low incidence and/or in the absence of a dose-response relationship these findings were considered not to be toxicologically significant.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There were no treatment-related deaths during the study period. One female (no. 341) in Group 2 was sent to necropsy on Day 4 of lactation because the only surviving pup of this dam was killed in extremis due to a poor condition.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects on body weights were noted up to 15000 ppm. At start of the pre-mating phase, Group 2-4 animals (both sexes) showed lower mean body weights as compared to animals of the control Group 1 (only statistically significant for Group 4 females). This trend continued, reaching statistical significance in males at 15000 ppm (Group 4) on pre-mating Days 8-29. In addition, a trend towards higher body weight gains was recorded for all treated groups (both sexes) as compared to control animals during pre-mating (only statistically significant for Group 4 females). These changes in body weight and body weight gain were caused by the fact that a few pups selected for Groups 2-4 were 8-11 days younger than the youngest pups selected for Group 1. Due to their younger age, these pups had a lower initial body weight and showed a higher body weight gain as compared to the older ones during the first weeks of pre-mating. At the end of the pre-mating period mean body weights were normalized in all treated groups.
The higher body weight gain noted in females at 1500 ppm (Group 2) on post-coitum Day 20 was considered not to be a treatment-related effect. The statistical significance was most likely caused by the fact that the mean body weight gain recorded for control animals was relatively low.
The higher body weight gain noted in males at 5000 ppm (Group 3) on mating Day 15 was considered to be a fortuitous finding since the change as compared to controls was very slight and no dose-effect relationship could be established.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically significant changes in food intake (absolute and relative to body weight) were noted up to 15000 ppm.
The slightly higher mean food intake (before and after allowance for body weight) noted in animals of Groups 2-4 (both sexes) on several occasions was not considered toxicologically relevant as values remained within the normal range. Moreover, if toxicity would be present, a reduction in food consumption would be expected instead.
At the individual level, absolute food consumption was relatively low for males in cage no. 17 (5000 ppm), especially in the first week of pre-mating. This lower mean food intake was most likely due to the fact that two out of four cage mates (nos. 265 and 266) had a relatively low intial body weight and thus consumed less food.

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):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no changes in organ weights that were considered to be of toxicological significance. The following statistically significant changes between mean organ weights of treated and control animals were noted: increased adrenal weights in males of Group 2 (relative) and Group 3 (absolute and relative) and females of Group 2 (absolute), and increased liver weights in females of Group 2 (absolute and relative) and Group 4 (relative). These findings were considered not to be a sign of toxicity, since no dose-response relationship could be established and/or all organ weights remained within the normal range of biological variation seen for animals of the same age and strain used in this type of study.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no macroscopic findings that were considered attributable to treatment with TERRACESS P.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related microscopic findings indicative of toxicity were identified in the reproductive organs of the F1-generation. All microscopic findings recorded were considered to be within the range of background pathology encountered in Wistar Han rats of these ages.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no treatment related effects on estrous cycle duration or classification. The percentage of females per group that were classified as having a ‘regular’ cycle was 87.5, 95.8, 95.8 and 87.5 % at 0, 1500, 5000 and 15000 ppm, respectively. In these animals, the incidence of irregular cycle was 12.5, 4.2, 4.2 and 12.5%, respectively.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed on spermatogenesis endpoints in F1-males. These spermatogenesis endpoints included mean testicular and epididymal sperm concentrations,
morphology (Groups 1 and 4), motility and progressive motility (all groups) The statistically significant changes noted in high dose males (Group 4) for epididymal sperm concentration, left testis weight and sperm morphology were not considered to be a sign of toxicity, as changes were small, remained within the normal range of biological variation seen for animals of the same age and strain used in this type of study and/or represented an improvement rather than deterioration.

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproduction parameters were unaffected by treatment up to 15000 ppm TERRACESS P. Mating, fertility, precoital time, conception rate, and number of implantation sites were comparable between treated and control animals.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted. Incidental clinical symptoms consisted of small size, chromodacryorrhoea left eye, tail apex bent or missing, wounds, (small) blue spots, alopecia and/or scabbing of several body parts. No
relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain. For clinical signs of pup 11 of litter 341 (Group 2) and pup 1 of litter 387 (Group 4), which were killed in extremis, see paragraph mortality in this section.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No treatment-related mortality occurred.
The number of pups (litters) which were found dead, missing or killed in extremis during lactation period was 2 (2), 15 (4), 1 (1) and 4 (4) for Groups 1, 2, 3 and 4, respectively. The relatively high mortality in Group 2 (1500 ppm) was due to one litter (no. 341). In this litter, eleven pups were recorded dead at first litter check. The only surviving pup (no. 11, female) was in a poor condition (i.e. cold appearance, (very) small size, no milk in the stomach, relatively low body weight on lactation Days 1 and 4, no body weight gain). Since the condition of the latter pup did not improve, it was decided on lactation Day 4 to kill the pup (with its dam) for humane reasons. At necropsy, isolated reddish foci on the thymus (dam) and no milk in the stomach
(pup) were noted. Since the high mortality in litter no. 341 was an isolated case without any dose-response relationship, it was considered to be a fortuitous finding with no toxicological relevance.

In Group 4, one pup (no. 5, female) in litter no. 389 died spontaneously on lactation Day 12. For this pup, no clinical signs were noted in the period before its death and body weights were within the normal range of biological variation. Necropsy findings included no milk and wound abdominal region. The latter observation could not be attributed directly to cannibalism. As all other littermates developed normal, this isolated death was considered a fortuitous finding. In
the same Group 4, one pup (no. 1, male) in litter no. 387 was killed in extremis on lactation Day 17. For this pup small size, large head, abnormal eyes and/or lethargy were recorded in the week before sacrifice. Necropsy findings included small size, abnormal eyes, brain cavity formation, and enlarged brain with liquid (latter observation not shown in tables). Hydrocephalus is occasionally seen among rats used in these types of studies. Moreover, all other littermates
developed normal. Therefore, this isolated case was considered not to be related to treatment.

For female no. 298 in Group 1, delivery of one pup was noted on December 26, 2009. However, at first litter check no pups were found. Therefore, no data on this pup were available. As female no. 298 was a control animal, no biological significance was attached to this observation. Due to the absence of pups at first litter check, it was decided to exclude this animal from lactation phase.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment up to 15000 ppm did not affect body weights of the pups. At the individual level, relatively low body weights were recorded for one pup in litters 312 (M4, Group 1), 335 (M8, Group 2), 380 (M1, Group 4) and 387 (M1, Group 4) each on one or more occasions. In line with this, small size was noted during the daily observations and/or at necropsy. At the low incidence and due to the fact that control as well as treated animals were affected, this finding was not considered to be toxicologically-relevant.

Food consumption and compound intake (if feeding study):

not examined

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:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

F1-PUP DEVELOPMENT
No treatment-related effects on postnatal pup development (dead and living pups at first litter check, sex ratio, postnatal loss (until lactation Day 4), breeding loss (lactation Day 5-21), viability index and weaning index, clinical signs, body weight, vaginal opening and balanopreputial separation, macroscopy and organ weights) were noted.

F1-PUP mortality:
No treatment-related mortality occurred.

The number of pups (litters) which were found dead or missing during the first days of lactation was 3 (3), 7 (3), 1 (1) and 1 (1) for Groups 1, 2, 3 and 4, respectively. The relatively high mortality in Group 2 (1500 ppm) was due to one litter (no. 136). In this litter of eleven pups, two pups were recorded dead at first litter check and three more pups were recorded missing on lactation Day 2. At this low incidence and in absence of a dose-response relationship, these deaths were not considered to be related to treatment.

F1-PUP clinical signs:
No treatment-related clinical signs were noted.

Incidental clinical symptoms consisted of small size, small eye (not confirmed at necropsy), pale and cold appearance, no milk in the stomach, blue discolouration of snout or skin around the left eye, blue spots on head or neck, and alopecia and/or scabbing of several body parts. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

F1-PUP body weights:
Treatment up to 15000 ppm did not affect body weights of the pups.

At the individual level, relatively low body weights were recorded for one pup in one litter in Group 1, one in Group 2 and two litters in Group 4 each on one or more occasions. In line with this, small size was noted during the daily observations and/or at necropsy (except for the pup in the litter in Group 1). At the low incidence and due to the fact that control as well as treated animals were affected, this finding was not considered to be toxicologically relevant.

F1-PUP Vaginal opening and balanopreputial separation:
There were no differences in age or weight upon reaching vaginal patency or balanopreputial separation following treatment with TERRACESS P up to 15000 ppm.

The statistically significantly higher age and weight upon reaching balanopreputial separation recorded for Group 2 males (1500 ppm) was considered to have occurred by chance, since no dose-effect relationship could be established.

F1-PUP Macroscopic findings:
No treatment-related macroscopic findings were noted up to 15000 ppm.

No milk in the stomach and autolysis were commonly noted for animals that were found dead. In addition, for one pup found dead cannibalism of the nose was recorded.

Incidental findings included small size, pelvic dilation of the kidney, enlarged spleen, and dilation of a brain ventricle. Due to the low incidence they were considered to have occurred by chance.

F1-PUP Organ weights:
Treatment up to 15000 ppm did not affect organ weights of the pups.


F2-PUP DEVELOPMENT
F2-PUP Mortality:
No treatment-related mortality occurred.

The number of pups (litters) which were found dead, missing or killed in extremis during lactation period was 2 (2), 15 (4), 1 (1) and 4 (4) for Groups 1, 2, 3 and 4, respectively.

The relatively high mortality in Group 2 (1500 ppm) was due to one litter. In this litter, eleven pups were recorded dead at first litter check. The only surviving female pup was in a poor condition (i.e. cold appearance, (very) small size, no milk in the stomach, relatively low body weight on lactation Days 1 and 4, no body weight gain). Since the condition of the latter pup did not improve, it was decided on lactation Day 4 to kill the pup (with its dam) for humane reasons. At necropsy, isolated reddish foci on the thymus (dam) and no milk in the stomach (pup) were noted. Since the high mortality in this litter was an isolated case without any dose-response relationship, it was considered to be a fortuitous finding with no toxicological relevance.

In Group 4, one female pup died spontaneously on lactation Day 12. For this pup, no clinical signs were noted in the period before its death and body weights were within the normal range of biological variation. Necropsy findings included no milk and wound abdominal region. The latter observation could not be attributed directly to cannibalism. As all other littermates developed normal, this isolated death was considered a fortuitous finding. In the same Group 4, one male pup was killed in extremis on lactation Day 17. For this pup small size, large head, abnormal eyes and/or lethargy were recorded in the week before sacrifice. Necropsy findings included small size, abnormal eyes, brain cavity formation, and enlarged brain with liquid (latter observation not shown in tables). Hydrocephalus is occasionally seen among rats used in these types of studies. Moreover, all other littermates developed normal. Therefore, this isolated case was considered not to be related to treatment.

For one female in Group 1, delivery of one pup was noted on December 26, 2009. However, at first litter check no pups were found. Therefore, no data on this pup were available. As this female was a control animal, no biological significance was attached to this observation. Due to the absence of pups at first litter check, it was decided to exclude this animal from lactation phase.

F2-PUP Clinical signs:
No treatment-related clinical signs were noted.

Incidental clinical symptoms consisted of small size, chromodacryorrhoea left eye, tail apex bent or missing, wounds, (small) blue spots, alopecia and/or scabbing of several body parts. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

For clinical signs of one pup of a litter in Group 2 and one pup of a litter in Group 4, which were killed in extremis, see section F2-PUP Mortality.

F2-PUP Body weights:
Treatment up to 15000 ppm did not affect body weights of the pups.

At the individual level, relatively low body weights were recorded for one pup in one litter in Group 1, one in Group 2 and two in Group 4 each on one or more occasions. In line with this, small size was noted during the daily observations and/or at necropsy. At the low incidence and due to the fact that control as well as treated animals were affected, this finding was not considered to be toxicologically-relevant.

F2-PUP Macroscopic findings:
No treatment-related macroscopic findings were noted up to 15000 ppm.

For one male pup each in two litters in Group 2 situs inversus was recorded at necropsy. Based on body weights, both pups had developed normally. No clinical signs were noted. As situs inversus is occasionally seen in rats of this strain and no dose-response relationship could be established, it was considered to be a fortuitous finding.

For necropsy findings of one pup in a litter in Group 2 and one pup in a litter in Group 4, which were killed in extremis, see section F2-PUP Mortality.

No milk in the stomach and autolysis were commonly noted for animals that were found dead. In addition, for one pup found dead cannibalism was recorded.

Incidental findings included small size, wound on the right foreleg, chromodacryorrhoea left eye, right eye reduced in size, tail apex bent or missing, and right kidney enlarged and gelatinous. These findings were considered to be of no toxicological relevance, as they occurred at a very low incidence and/or no relationship with treatment could be established.

F2-PUP Organ weights:
Treatment up to 15000 ppm did not affect organ weights of the pups.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 15 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 15 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity observed at highest dose tested.

Key result

Reproductive effects observed:

no

Conclusions:

In this two-generation study, TERRACESS P was administered via the diet through two complete reproductive cycles to SPF-bred male and female Wistar Han rats. The dose levels tested were 1500, 5000 and 15000 ppm.
Analysis of the diet preparations used in this study revealed acceptable accuracy and homogeneity.

Parental results
No parental toxicity was observed at any dose level.
There were no treatment-related deaths or clinical signs. No adverse effects were noted on body weight, body weight gain, food consumption, macroscopy and organ weights following treatment with TERRACESS P up to 15000 ppm. There were no treatment-related microscopic findings indicative of toxicity or responsible for suspected infertility in the reproductive organs in either F0- or F1-generation.

There was no adverse effect of treatment with the test item on the number of primordial follicles in the ovaries of the sampled F1-females.
No changes in estrous cycle or spermatogenesis endpoints were observed in either generation up to 15000 ppm.

Reproductive/Develomental results
Reproduction parameters (mating, fertility, precoital time, conception rate, and number of implantation sites) were comparable between treated and control animals. Gestation index and duration were comparable between treated and control animals. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed. No treatment-related effects on postnatal pup development (dead and living pups at first litter check, sex ratio, postnatal loss, breeding loss, viability index and weaning index, clinical signs, body weight, age and weight upon reaching vaginal patency or balanopreputial separation (only determined for F1-pups), macroscopy and organ weights) were noted in either generation.

Treatment of male and female Wistar Han rats at dose levels of 1500, 5000 and 15000 ppm TERRACESS P revealed neither parental toxicity nor effects on reproduction and development.

Based on these findings, the parental, reproduction and development No Observed Adverse Effect Levels (NOAEL) were established as being at least 15000 ppm.

When corrected for mean test article intake the NOAEL of 15000 ppm corresponds to 930-1239 mg and 1183-2679 mg TERRACESS P per kg body weight per day for males and females, respectively.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A two-generation reproduction study with Terracess P was performed according to OECD, EPA and EC test guidelines, in rats by dietary administration. Dose levels were selected based on results of a dose range finding study. After acclimatisation, four groups of twenty-four male and twenty-four female Wistar Han rats were exposed by dietary administration to the test substance at 0, 1500, 5000, 15000 ppm. The F0-generation was exposed for a minimum of 70 days prior to mating and continuing until euthanasia. The F1-generation was potentially exposed to the test substance in utero, through nursing during lactation and directly following weaning. After weaning, pups were treated for a minimum of 70 days prior to mating and continuing until euthanasia. The F2-generation was

potentially exposed to the test substance in utero and through nursing during lactation. The following parameters were evaluated: mortality, clinical signs, body weights, food consumption, reproduction processes, observations on offspring, macroscopy, organ weights, and histopathology. Chemical analyses of diets were conducted six times to assess accuracy and homogeneity.

Analysis of the diet preparations used in this study revealed acceptable accuracy and homogeneity. In both F0- and F1-generations there were no signs of parental, reproduction and developmental toxicity up to 15000 ppm. Treatment of male and female Wistar Han rats at dose levels of 1500, 5000 and 15000 ppm Terracess P revealed neither parental toxicity nor effects on reproduction and development. Based on these findings, the parental, reproduction and development NOAELs were established as being at least 15000 ppm. When corrected for mean test article intake the NOAEL of 15000 ppm corresponds to 930 -1239 mg and 1183-2679 mg TERRACESS P per kg body weight per day for males and females, respectively.

Short description of key information:

In a 2-generation reproduction toxicity study with rats treated orally with Terracess P did not show any adverse effects up to the highest dose tested, 1000 mg/kg bw/day neither on the parental animals, nor on the pups.

Justification for selection of Effect on fertility via oral route:

This test was performed with Terracess P, a compound which has been demonstrated to be very similar in structure, physicochemical properties and toxicological profile to Terracess TF in the Read across Justification document for reprotoxicity and mutagenicity (see section 13). Due to the fact Terracess TF and Terracess P have nearly the same chemical structure, the same interaction with bio-molecules, living cells and tissue is expected. Therefore, a read across from Terracess TF to data obtained with Terracess P is scientifically justified.

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study with Terracess P in rats by oral gavage performed according to current test guidelines (OECD, EC, EPA), showed a NOAEL of at least 1000 mg/kg bw/day as no adverse treatment related effects were observed.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-06-2009 to 03-07-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: approximately 14 weeks.
- Weight at study initiation: 199-261 grams (day 0 post-coitum)
- Fasting period before study: not applicable
- Housing:
Pre-mating During acclimatization, females were housed in groups of 5 animals/cage in
Macrolon cages (MIV type, height 18 cm). During the weekend, mating procedures were suspended and the animals were housed in groups of maximum 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon
cages (MIII type, height 18 cm).
Post-coitum Females were individually housed in Macrolon cages (MIII type, height 18 cm).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to the start of treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4-21.3
- Humidity (%): 34-79
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 02 June 2009 (start pairing) To: 23 June 2009

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity of the test substance, vehicle, and/or formulation.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis (for accuracy and homogeneity) was done on one day during the treatment period (09 June 2009) according to a validated method (NOTOX Project 490988). The analytical method for the quantitative analysis of the test substance in formulation is based on total Titanium analysis after acidic digestion. During this digestion, the test substance is split into individual ions. The test substance cannot be analysed in its original form. Stability of the test substance in formulations could therefore not be tested.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: [yes ]
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
-After successful mating each pregnant female was caged: individually
-Any other deviations from standard protocol: no

Duration of treatment / exposure:

From day 6 to day 19 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Duration of test:

02 June 2009 (start pairing) to 03 July 2009 (end of in-life phase).

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on results of the dose range finding study (NOTOX Project 490450). Four groups of 6 females were exposed to 100, 300 and 1000 mg/kg/day for Days 6 to 19 post-coitum inclusive by oral gavage. There were no signs of toxicity.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations: Mortality/Viability


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 0 post-coitum onwards


BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3 and 6-20 (daily) post-coitum.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, determined on Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.



WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No



POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: Day 20 post-coitum
- Organs examined: External, thoracic and abdominal organs

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett,
1955) (many-to-one t-test) based on a pooled variance estimate was applied for the
comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be
assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.
Mean litter proportions (percent per litter) of total fetal malformations and developmental
variations (external, visceral, skeletal and combined), and each particular external, visceral and
skeletal malformation or variation was subjected to the Kruskal-Wallis nonparametric ANOVA
test (Kruskal and Wallis 1952) to determine intergroup differences. If the ANOVA revealed
statistically significant (p<0.05) intergroup variance, Dunn’s test (Dunn 1964) was used to
compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of
significance. Group means were calculated for continuous data and medians were calculated for
discrete data (scores) in the summary tables. Test statistics were calculated on the basis of
exact values for means and pooled variances.

Indices:

For each dose group reproduction parameters were expressed in two ways:
-As a mean (with standard deviation) of the number observed for each litter
-As a mean litter proportion calculated on a total group basis
For each litter the following calculations were performed:
Pre-implantation loss = (Number of corpora lutea - number of implantation sites) / Number of corpora lutea x 100
Post -implantation loss = (Number of implantation sites - number of live fetuses) / Number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given
finding both as the number of fetuses and the number of litters available for examination in the
group; and 2) considering the litter as the basic unit for comparison, calculating the number of
affected fetuses as a mean litter proportion on a total group basis.
Where:
Viable Fetuses Affected/Litter (%) = ( No. Viable Fetuses Affected/Litter) / No. Viable Fetuses/Litter x 100

Historical control data:

NOTOX Historical control data were used for external, visceral and skeletal malformations.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Animal 42, Group 2 (100 mg/kg) had hunched posture, ptosis, piloerection, hypothermia and lethargy on Day 19 post coitum and was killed in extremis (see 7.2.1. Mortality above). Due to the isolated nature of these findings, seen only in one animal on a single day of the treatment period, they were likely due to chance and not indicative of treatment-related toxicity.
Incidental findings included alopecia of various body parts. This is not uncommonly seen in animals of this age and strain, and does not indicate treatment-related toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Animal 42, Group 2 (100 mg/kg) was killed in extremis on Day 19 post coitum. Dark red discoloration and hardening of the uterus and cervical torsion were noted at the macroscopic examination; as this was an isolated finding, and in the absence of any dose-related relationship, these findings were not indicative of treatment-related toxicity.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on body weights (before or after correction for gravid uterine weights) or % of body weight gain with treatment of TERRACESS P up to 1000 mg/kg.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Absolute and relative food consumption were unaffected with treatment of TERRACESS P up to 1000 mg/kg.

Food efficiency:

not examined

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):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance related effects on macroscopic findings at any dose level. Incidental findings included alopecia of various body parts. A single female, animal 42 (100
mg/kg) was killed in extremis on Day 19 of post coitum; see the Mortality section for the macroscopic findings noted for this animal.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

effects observed, non-treatment-related

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no toxic effects observed.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

mortality

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

maternal abnormalities

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean fetal body weights were 3.6, 3.5, 3.5 and 3.6 grams for Groups 1, 2, 3 and 4, respectively.

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Male:female sex ratios were comparable between controls and all treatment groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Mean litter sizes were 11.0, 11.5, 11.9 and 10.5 fetuses for Groups 1, 2, 3 and 4, respectively.

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on fetal external morphology. Fetus no. A094-01 in the 1000 mg/kg group had a cyst-like growth from a cleft in the anterior palate into the buccal cavity. At visceral cephalic examination this fetus also showed anophthalmia (the right eye was absent) and misshapen brain. The isolated nature of these findings did not indicate any association with test substance treatment. No external developmental malformations or variations were observed in any other fetuses in this study. One fetus in the 100 mg/kg group (no. A045-02) and one fetus at 1000 mg/kg (no. A083-04) had their placenta grown together with an adjacent placenta. This finding was considered a pathological alteration and not a developmental alteration, and therefore was not classified as
either a malformation or a developmental variation and was not included in the summary tables, but was included in the individual tables.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on fetal skeletal morphology. Control fetus A024-02 had fused skull bones (the jugal was fused to the maxilla, unilateral). No other skeletal malformations were noted in this study. The mean litter proportion of bent ribs was 10.0%, 4.0%, 1.0% and 6.1% in the control, 100, 300 and 1000 mg/kg groups, respectively. At 300 mg/kg, the mean litter proportion of bent ribs was significantly lower than in the control group and lower than the minimum historical control value (historical control data range: 6.7% - 15.2%). However, a decrease in the number of fetuses
with bent ribs is not an adverse effect and the group distribution for this finding does not suggest a relation to test substance treatment. Therefore, the decreased number of fetuses with bent ribs at 300 mg/kg was considered to have arisen by chance.
Other skeletal developmental variations observed in the test substance groups were 14th rudimentary ribs, ossified cervical centrum no. 1, slightly to moderately malaligned sternebra(e), reduced ossification of the skull, 7th cervical rib(s), unossified metacarpals and metatarsals, unossified sternebra(e), 14th full ribs, 27 presacral vertebrae, generalized reduced ossification of the entire skeleton, unossified hyoid, unco-ossified and unossified vertebral centra. All these variations occurred at similar frequencies in the control group, occurred infrequently and/or in a manner that was not dose-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on fetal visceral morphology.
Fetus no. A078-01 in the 1000 mg/kg group had external hydrocephaly (increase in space between the brain and the dura mater) and fetus A034-02 at 100 mg/kg had microphthalmia (left eye). Because each finding occurred in single fetuses and was seen in NOTOX historical controls (hydrocephaly) or occurred in the lowest dose group (microphthalmia), they were not considered related to the test substance.
No other visceral malformations were observed in this study. Visceral developmental variations observed in the test substance groups were accessory liver lobules, hemorrhagic adrenals, pale spleen and accessory spleen. All these variations occurred at similar frequencies in the control group and/or occurred infrequently.
One control fetus (no. A007-16) had a red area on the heart. This finding was considered a pathological alteration and not developmental alteration, and therefore was not classified as
either a malformation or a developmental variation and was not included in the summary tables, but was included in the individual tables.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no toxic effects observed.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: cranium

external: ear

external: eye

external: face

external: limb

external: paw

external: tail

external: trunk

external: anogenital distance

external: anus

external: genital tubercle

external: large intestine

external: thorax

external: umbilicus

external: pelvic region

skeletal: skull

skeletal: skull, fontanelles

skeletal: skull sutures

skeletal: clavicle

skeletal: scapule

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

skeletal: pelvic girdle

skeletal: hindlimb

visceral/soft tissue: integumentary

visceral/soft tissue: gastrointestinal tract

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

visceral/soft tissue: heamatopoietic

visceral/soft tissue: immune system

visceral/soft tissue: musculoskeletal system

visceral/soft tissue: nervous system

visceral/soft tissue: central nervous system

visceral/soft tissue: peripheral nervous system

visceral/soft tissue: somatic nervous system

visceral/soft tissue: autonomic nervous system

visceral/soft tissue: endocrine system

visceral/soft tissue: respiratory system

visceral/soft tissue: male reproductive system

visceral/soft tissue: female reproductive system

visceral/soft tissue: eye

visceral/soft tissue: ear

Key result

Developmental effects observed:

no

Accuracy and homogeneity of formulations were demonstrated by analyses.

Maternal effects noted

Mortality:

Animal 42, Group 2 (100 mg/kg) was killed in extremis on Day 19 post coitum. Dark red discoloration and hardening of the uterus and cervical torsion were noted at the macroscopic examination; as this was an isolated finding, and in the absence of any dose-related relationship, these findings were not indicative of treatment-related toxicity.

Clinical signs:

Animal 42, Group 2 (100 mg/kg) had hunched posture, ptosis, piloerection, hypothermia and lethargy on Day 19 post coitum and was killed in extremis (see 7.2.1. Mortality above). Due to the isolated nature of these findings, seen only in one animal on a single day of the treatment period, they were likely due to chance and not indicative of treatment-related toxicity.

Incidental findings included alopecia of various body parts. This is not uncommonly seen in animals of this age and strain, and does not indicate treatment-related toxicity.

Fetal effects noted, external malformations and variations:

There were no test substance-related effects on fetal external morphology.

One fetus in the 1000 mg/kg group had a cyst-like growth from a cleft in the anterior palate into the buccal cavity. At visceral cephalic examination this fetus also showed anophthalmia (the right eye was absent) and misshapen brain. The isolated nature of these findings did not indicate any association with test substance treatment.

No external developmental malformations or variations were observed in any other fetuses in this study.

One fetus in the 100 mg/kg group and one fetus at 1000 mg/kg had their placenta grown together with an adjacent placenta. This finding was considered a pathological alteration and not a developmental alteration, and therefore was not classified as either a malformation or a developmental variation and was not included in the summary tables, but was included in the individual tables.

Visceral malformations and variations:

There were no test substance-related effects on fetal visceral morphology.

One fetus in the 1000 mg/kg group had external hydrocephaly (increase in space between the brain and the dura mater) and one fetus at 100 mg/kg had microphthalmia (left eye). Because each finding occurred in single fetuses and was seen in historical controls (hydrocephaly) or occurred in the lowest dose group (microphthalmia), they were not considered related to the test substance.

No other visceral malformations were observed in this study.

Visceral developmental variations observed in the test substance groups were accessory liver lobules, hemorrhagic adrenals, pale spleen and accessory spleen. All these variations occurred at similar frequencies in the control group and/or occurred infrequently.

One control fetus had a red area on the heart. This finding was considered a pathological alteration and not developmental alteration, and therefore was not classified as either a malformation or a developmental variation.

Skeletal malformations and variations:

There were no test substance-related effects on fetal skeletal morphology.

One control fetus had fused skull bones (the jugal was fused to the maxilla, unilateral). No other skeletal malformations were noted in this study. 

The mean litter proportion of bent ribs was 10.0%, 4.0%, 1.0% and 6.1% in the control, 100, 300 and 1000 mg/kg groups, respectively. At 300 mg/kg, the mean litter proportion of bent ribs was significantly lower than in the control group and lower than the minimum historical control value (historical control data range: 6.7% - 15.2%). However, a decrease in the number of fetuses with bent ribs is not an adverse effect and the group distribution for this finding does not suggest a relation to test substance treatment. Therefore, the decreased number of fetuses with bent ribs at 300 mg/kg was considered to have arisen by chance.

Other skeletal developmental variations observed in the test substance groups were 14^thrudimentary ribs, ossified cervical centrum no. 1, slightly to moderately malaligned sternebra(e), reduced ossification of the skull, 7^thcervical rib(s), unossified metacarpals and metatarsals, unossified sternebra(e), 14^thfull ribs, 27 presacral vertebrae, generalized reduced ossification of the entire skeleton, unossified hyoid, unco-ossified and unossified vertebral centra. All these variations occurred at similar frequencies in the control group, occurred infrequently and/or in a manner that was not dose-related.

Conclusions:

Mated female Wistar Han rats were assigned to four dose groups, each containing twenty-four animals. The test item was administered once daily by gavage from Day 6 to 19 post-coitum at doses of 100, 300 and 1000 mg/kg/day (Groups 2, 3 and 4 respectively). The rats of the control group received the vehicle, 1% aqueous carboxymethyl cellulose, alone. Accuracy and homogeneity of formulations were demonstrated by analyses.

Maternal findings
No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.

Developmental findings
No developmental toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for TERRACESS P was established as being at least 1000 mg/kg body weight/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A prenatal developmental toxicity study with Terracess P was performed according to OECD, EC and EPA test guidelines, in rats by oral gavage. Mated female Wistar Han rats were assigned to four dose groups, each containing twenty-four animals. The test item was administered once daily by gavage from Day 6 to 19 post-coitum at doses of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, 1% aqueous carboxymethyl cellulose, alone. Accuracy and homogeneity of formulations were demonstrated by analyses. Maternal findings: No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups. Developmental findings: No developmental toxicity was observed in the 100, 300 and 1000 mg/kg/day groups. In conclusion, based on the results in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Terracess P was established as being at least 1000 mg/kg body weight/day.

Justification for selection of Effect on developmental toxicity: via oral route:

This test was performed with Terracess P, a compound which has been demonstrated to be very similar in structure, physicochemical properties and toxicological profile to Terracess TF in the Read across Justification document for reprotoxicity, mutagenicity document (see section 13). Due to the fact Terracess TF and Terracess P have nearly the same chemical structure, the same interaction with bio-molecules, living cells and tissue is expected. Therefore, a read across from Terracess TF to data obtained with Terracess P is scientifically justified.

Justification for classification or non-classification

Terracess TF does not have to be classified for reproduction, fertility and developmental toxicity according to CLP Regulation EC (No.) 1272/2008, based on the available studies performed with its analogue, Terracess P.