Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: extended one-generation study
Type of information:
experimental study planned
Study period:
Within 40 months after issue of ECHA final decision (range-finder plus actual study)
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS


NON-CONFIDENTIAL NAME OF SUBSTANCE: Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene



CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:


•available GLP studies

Available GLP studies are part of the registration dossiers. None of them fulfil the requirements of an extended one-generation study.

•available non-GLP studies

Available Non-GLP studies are part of the registration dossiers. None of them fulfil the requirements of an extended one-generation study.


•historical human data
Databases containing potential information reproductive toxicity of the substance in published and internal data on the substance were searched and no contributing information was found.

•(Q)SAR
There are no reliable QSAR models addressing the endpoint of reproductive toxicity to the extent that is assessed in the extended one-generation study.

•in vitro methods
There are no reliable in-vitro methods addressing the endpoint of reproductive toxicity to the extent that is tested in the extended one-generation study.


•weight of evidence: Considering the lack of in-vitro methods, QSAR models and multigeneration studies on the substance itself and potential analogues, a weight-of-evidence assessment is not possible.

•Grouping and read-across: Potential candidates for grouping and read-across can be found in the OECD IATA case study document on the endpoint of repeated-dose toxicity. Also a list of potential candidates can be found in the Canadian draft screening risk assessment on the category of substituted alkylated diphenylamines. Since none of the potential candidates has been tested for reproductive toxicity in the extended one-generation study, read-across is not possible. For details^ it is refered to the table in the attachment.

•substance-tailored exposure driven testing: not applicable

•approaches in addition to above: not applicable

•other reasons: not applicable

•Considerations that the specific adaptation possibilities of Annexes VI to X (and column 2 thereof) were not applicable: Based on the existing data and hazard and use profile of the substance in question, it is not possible to apply specific adaptation possibilities listed in Annexes IX and X.

Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
Modules for DNT and DIT are not considered relevant for this substance. The need for mating of the F1 generation will be determined in the course of the study.
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Route of administration:
oral: feed
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no study available (further information necessary)
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

Information is available at the screening level (OECD 422) with doses of 225, 75 and 25 mg/kg bw (BASF 2014). No toxicologically relevant effects on reproductive parameters were noted with treatment up to 225 mg/kg bw/day. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. There were 10, 10, 10 and 9 pregnant females in the control, 25, 75 and 225 mg/kg bw/day groups, respectively.


Short description of key information:
Information is available at the screening level (OECD 422) with doses of 225, 75 and 25 mg/kg bw. Adverse effects on liver as indicated by changes in clinical chemistry, gross necropsy, histopathology and absolute and relative weights occurred at the mid and high dose groups. No adverse effects on fertility were observed. A testing proposal for a 2-generation study was submitted in 2010. The final decision for this study is pending (status June 26, 2015).

Effects on developmental toxicity

Description of key information
Information is available at the screening level (OECD 422) with doses of 225, 75 and 25 mg/kg bw. Adverse effects on liver as indicated by changes in clinical chemistry, gross necropsy, histopathology and absolute and relative weights occurred at the mid and high dose groups. At the high dose group, there was an increased incidence of postnatal mortality which occurred mostly in one litter and is unlikely to represent true developmental toxicity.  The final decision on a testing proposal for a two-generation study is pending. Regarding teratogenicity, an OECD 414 study in rats with the structural analogue Benzeneamine, N-phenyl, reaction products with nonene, branched are available. This study was performed with doses of 50, 150 and 500 mg/kg bw.  No teratogenicity and no embryotoxicity were observed. The high dose caused maternal toxicity.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD 414 and GLP compliant study. A default reliability of 2 is assigned for read-across according to ECHA guidance.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: (age at delivery 10 weeks)
- Weight at study initiation: (weight range at delivery 177-196 g)
- Fasting period before study: none
- Housing: individual cages (during gestation)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2014-05-20 To: 2014-06-16
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.


VEHICLE
- Justification for use and choice of vehicle: The substance is miscible in corn oil and insoluble in water.
- Concentration in vehicle: 12.5, 37.5 and 125 mg/mL
- Amount of vehicle: 4 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The proposed formulation procedure for the test item was checked in the range from 12.5 to 125 mg/mL by chemical analysis (concentration and
homogeneity) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits
for concentration (90-110%). Stability after 24 hours at room temperature was verified in the range from 1 to 300 mg/mL in the validation study.
Samples of the formulations prepared on week 1 and Last Week were analysed to check the homogeneity and concentration.
Details on mating procedure:
Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of matingwas made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum).
Duration of treatment / exposure:
Day 6 through Day 19 post coitum
Frequency of treatment:
daily
Duration of test:
Day 6 through Day 20 post coitum
Remarks:
Doses / Concentrations:
50, 150 and 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Range-finding study with pregnant rats. The highest dose group of 500 mg/kg was expected to cause maternal toxicity as indicated by adverse effects on body weights and evidence on liver toxicity as indicated by clinical chemistry parameters. For details it is referred to the robust study summary of the maternal toxicity study.
- Rationale for animal assignment: Females were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum

FOOD CONSUMPTION : Yes
- Time schedule for examinations: Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: ovaries and uteri

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: gross evaluation of placentae, number of intra-uterine deaths,. Uteri or individual uterine horns without visible implantations were immersed
in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the
homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences
between the control and treated groups assessed by a non-parametric version of the Williams test. The mean values, standard deviations and statistical
analysis were calculated from actual values in the computer without rounding off.
Indices:
Preimplantation loss
Postimplantation loss
Total implantation loss
Sex ratios
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
A slight decrease in body weight was noted in all treated females when compared to controls, reaching a statistical significance in females receiving
500 mg/kg bw/day (up to 7%), starting from Day 9 post coitum until the end of the study.
Statistically significant decrease was also recorded in body weight gain of females of the same group on Day 9 post coitum (109%; body weight loss) and Day 12 post coitum (22%). Starting from Day 15 post coitum the mean values of body weight gain were comparable between control and high dose group.

Statistically significant decrease (up to 22%) in food consumption was observed in treated females receiving 500 mg/kg bw/day, starting from Day 9 post coitum until the end of the study.

A slight trend to decrease was observed in terminal body weight of all treated females with respect to the control. This change was about -6% in the high
dose group, without statistical significance. A statistically significant decrease in corrected body weight (up to 6%) and corrected body weight gain (up
to 50%) was noted in treated females receiving 150 and 500 mg/kg bw/day. Gravid uterus weight was similar between control and treated groups.

There were no adverse findings at the macroscopic examination at necropsy.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter data, mean foetal weight and sex ratio were unaffected by treatment.

A total of 15 small foetuses (foetal weight < 2.7 g) were detected: 2 out of 269 in the control group, 2 out of 214 in the low dose group, 1 out of 269 in the mid-dose group and 10 out of 254 in the high dose group. One foetus in the high dose group showed malrotation of the hindlimb, considered incidental. Of the ten small fetuses in the high dose group, 7 were from the dam which suffered most strongly from maternal toxicity as indicated by the lowest corrected body weight gain of minus 9.8g. This dam was also the only dam showing hunched posture and piloerection on gestation day 20. The higher incidence of small foetuses is therefore considered to be related to the lower maternal body weight gain.

No relevant findings that could be considered treatment-related were observed at visceral examination of foetuses in the treated groups, compared to controls.
The alterations recorded at skeletal examinations of foetuses were noted both in control and treated groups with a similar incidence.
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Remarks on result:
other: No teratogenicity at the hightest tested dose
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: embryotoxicity
Remarks on result:
other: No embrotoxicity at the highest tested dose
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: TERMINAL BODY WEIGHT, UTERUS WEIGHT, CORRECTED BODY WEIGHT AND CORRECTED BODY WEIGHT GAIN OF FEMALES - GROUP MEAN DATA

Group   Terminal body weight (g) Gravid uterus weight (g) Body weight at necropsy minus gravid uterus weight (corrected body weight) (g)  Body weight at necropsy minus gravid uterus weight, minus body weight on GD6 (corrected body weight gain) (g) 
1 (control) Mean 336.38 66.40 269.98 29.87
  SD 15.84 8.80 14.79 7.67
  (n) 23 23 23 23
2 Me 323.9 60.42 263.5 29.45
  SD 23 18 18 16
  (n) 20 20 20 20
3 Me 323.8 66.20 257.6* 20.5*
  SD 28 12 18 9.
  (n) 23 23 23 23
4 Me 315.9 62.53 253.3* 14.83*
  SD 24 14 23 16
  (n) 22 22 22  22

* = Statistically significantly different from control group value at p< 0.05

Table 2: FOOD CONSUMPTION (g/animal/day) - GROUP MEAN

Group   gestation day 3 gestation day 6 gestation day 9 gestation day 12 gestation day 15 gestation day 18 gestation day 20
1 (control) n  23 23 23 23 23 23 23
  Mean 18.13 20.66 18.72 20.99 21.78 23.68 23.06
  SD 2.37 2.54 2.05 1.98 2.10 2.20 1.99
2 n  20 20 20 20 20 20 20
  Mean 17.99 20.97 18.32 20.64 21.04 23.43 22.83
  SD 2.3 3.03 2.46 2.19 2.68 2.99 4.33
3 n  23 23 23 23 23 23 23
  Mean 18.7 20.23 17.36 19.28 20.90 22.32 21.56
  SD 1.99 2.53 2.59 2.90 2.40 2.91 3.14
4 n  22 22.00 22 22.00 22.00 22.00 22.00
  Mean 18.24 20.97 15.33** 17.1** 19.01** 20.12** 18.02**
  SD 2.62 2.55 1.98 2.76 2.68 3.02 2.96

** = mean value of group is significantly different from control at p < 0.01

Table 3: Pregnancy status overview

Group 1 (control) 2 3 4
Initial group size (n) 24 24 24 24
Not pregnant (n) 1 4 1 2
Unilateral implantation (n) 0 1 0 0
With live foetuses at gestation Day 20 (n) 23 20 23 22

Table 4: Litter data and sex ratios - group mean data

    Corpus lutea Implantations Early Uterine Deaths Late Uterine Deaths Total Uterine Deaths Viable Young (total) Viable males Viable females % Males Preimplantation loss (%) Postimplantation loss Total implantation loss(%) Litter weight (g) Mean fetal weight (g)
1 Mean 12.87 12.26 0.52 0.04 0.57 11.7 6 5.7 50.96 4.68 4.15 8.52 43.1 3.69
  SD 1.63 1.63 1.47 0.21 1.5 1.77 2.24 2.05 17.27 5.13 10.27 11.78 6.71 0.34
  (n) 23 23 23 23 23 23 23 23 23 23 23 23 23 23
2 Mean 12.26 11.47 0.21 0.05 0.26 11.21 5.67 5.84 49.82 6.56 4.12 10.05 39.88 3.63
  SD 2.58 2.44 0.54 0.23 0.56 2.84 2.22 2.29 14.18 6.13 11.67 13.68 9.32 0.4
  (n) 19 19 19 19 19 19 18 19 18 19 19 19 19 19
3 Mean 13.09 12.22 0.52 0 0.52 11.7 5.78 5.91 449.19 6.28 4.73 10.86 42.41 3.65
  SD 2.7 2.35 0.73 0 0.73 2.55 1.93 1.98 12.79 6.68 6.74 7.39 8.85 0.28
  (n) 23 23 23 23 23 23 23 23 23 23 23 23 23 23
4 Mean 12.27 11.73 0.14 0 0.14 11.59 5.36 6.23 46.95 6.11 1.18 7.23 40.11 3.51
  SD 2.66 3.06 0.35 0 0.35 3.08 2.06 2.25 12.97 10.6 3.07 10.96 9.86 0.39
  (n) 22 22 22 22 22 22 22 22 22 22 22 22 22 22
Conclusions:
The substance is not teratogenic and not embryotoxic in rats. It causes maternal toxicity at a dose level of 500 mg/kg bw.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available (further information necessary)
Quality of whole database:
There is a developemental toxicity screening study with the substance itself and a teratogenicity study with an analogue substance. There is currently a data gap for the second species; however the outcome of the extended one-generation study and the Canadian Chemical Management Plan draft risk assessment needs to be known prior to deciding on a testing proposal.
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

Information is available at the screening level (OECD 422) with doses of 225, 75 and 25 mg/kg bw (BASF 2014).

No toxicologically relevant effects on the gestation index and duration, parturition, maternal care or on most aspects of early postnatal pup development (clinical signs, body weight and macroscopy) were observed up to 225 mg/kg bw/day. The gestation index and duration of gestation were similar between all groups. Female no. 50 (Group 1) was euthanized in extremis after having a prolonged parturition. No other 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 and no deficiencies in maternal care were observed.

The number of dead pups at first litter check and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

At 225 mg/kg bw/day the mean number of living pups at first litter check (7.9) was significantly lower than for controls (10.9). Female no. 80 had only 4 pups, which contributed to this slightly low mean. Discounting her data, there were a mean of 8.4 pups/litter, which was also lower than controls and an effect of treatment could not be excluded.

The postnatal loss was also significantly higher for animals at 225 mg/kg bw/day with 11.3% of living pups lost (8 pups over 3 litters) than controls (no pups lost). High dose animals also had a correspondingly low viability index of 88.7%.

One pup was found dead at the first litter check and one pup was missing on postnatal Day 2 at 25 mg/kg bw/day. At 225 mg/kg bw/day, 7 pups (4 females, 3 males) went missing between Days 2 and 5 of lactation and 1 female pup was euthanized in extremis on lactation Day 1. Missing pups were most likely cannibalised. No pups died or went missing in the control and 75 mg/kg bw/day groups. Five of the missing pups at 225 mg/kg bw/day were from a single litter (no. 75; three other pups from this litter

survived to the end of the necropsy period), two were from another litter (no. 74). The majority of the pups came from a single litter and there were no signs of ill health in other pups from this dose level.

However, when taken together with the lower mean number of pups at first litter check, an effect of treatment could not be excluded. No toxicological relevance was attributed to the dead/missing pups at 25 mg/kg bw/day since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Clinical signs noted for pups who went missing, died or were euthanized in extremis included cold, no milk in the stomach, wounds (various body areas), pale and lean appearance. Incidental clinical signs noted for pups surviving to the scheduled necropsy period included scabs (various body areas) and pale appearance. The nature and incidence of these signs are not uncommon for pups of this age and strain and were not considered to be treatment related or toxicologically relevant. Pup body weights were unaffected by treatment up to 225 mg/kg bw/day.

Sore on the throat region was seen for the pup that was euthanized in extremis. This was the only macroscopic finding seen for pups that died before the scheduled necropsy. Scabbing on the chest was the only finding noted for pups surviving to the scheduled necropsy. These findings were both incidental in nature. Overall, other than the higher incidence of postnatal mortality, no adverse effects on offspring were observed. Considering the nature of a screening study and the high incidence in one single litter, this finding is not clearly attributable to treatment. Further clarification will be obtained once the results of the two-generation study have become available.

In addition, a developmental toxicity study in rats (OECD 414) with the branched nonylated analogue is available (BASF 2014b). Dose levels were 50, 150 and 500 mg/kg bw and corn oil was used as vehicle. Dose levels were chosen based on a range-finding study in pregnant rats with 300 and 1000 mg/kg bw (BASF Project No: 10R0227/12X519). In the maternal toxicity study, alterations of some liver/metabolic parameters were found specially in the high dose group and included increased values of alkaline phosphatase, alanine aminotransferase, cholesterol, triglycerides, glucose and decreases of creatinine, protein, albumin, globulin, calcium and bile acids. Relative liver weights were increased. Increased absolute and relative adrenals weights and decreased absolute spleen and kidneys weights were found in the high dose group when compared to controls. Values of alkaline phosphatase, triglycerides and bile acids were also different to controls at 300 mg/kg bw/day. Pale discoloration of liver was observed in four dams of the high dose group. Terminal body weight, corrected body weight and corrected body weight gain were significantly reduced in the high dose group. Uterus weight did not differ between groups.

The observed toxicity profile is consistent with that of the target substance. It is referred to both the toxicokinetic section and the endpoint summary for repeated dose toxicity for a detailed assessment of the read-across. The doses chosen for the main study are sufficient to ensure some but not excessive maternal toxicity in the main study.

In the main study, statistically significant decrease in body weight (up to 7%) and body weight gain was observed in treated females receiving 500 mg/kg bw/day, starting from Day 9 post coitum. Statistically significant decrease in food consumption (up to 22%) was observed in treated females receiving 500 mg/kg bw/day, starting from Day 9 post coitum. Statistically significant decrease in corrected body weight and corrected body weight gain was noted in treated females receiving 150 and 500 mg/kg bw/day. The difference at 150 mg/kg bw/day was minor (258 versus 270 g) and therefore not considered adverse. There were no gross findings at necropsy. Considering the effects on food consumption, corrected body weight and corrected body weight gain and the knowledge on clinical chemistry from the range-finding study, the high dose group caused liver toxicity and the NOAEL for maternal toxicity was 150 mg/kg. Litter data, mean foetal weight and sex ratio were unaffected by treatment. No treatment-related differences were seen at visceral examination of foetuses between the control and treated groups. No relevant changes were recorded at the skeletal examination of foetuses in treated groups, compared to controls. An increase in the presence of small foetuses was recorded in the high dose group. This higher number of small foetuses was due to one litter, and the corresponding

dam had the smallest corrected body weight gain and was most strongly affected by maternal toxicity. Overall, no teratogenic and embryotoxic hazard was identified.

Justification for classification or non-classification

The available experimental data does meet the criteria laid out for classification and labelling in EC Regulation 1272/2008

In the absence of results of the two-generation-study, no final assessment is possible.