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Description of key information

Regarding the repeated-dose toxicity of ‘Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol' (OAPP) (previous name: phenol, methylstyrenated), one oral subchronic study (100d) and two subacute studies (28d), an oral and a dermal one, are available:

1.) In the oral medium-term repeated dose toxicity study (OECD TG 408 within OECD 443, combined EOGRTS), repeated oral dietary exposure to approximately 122 mg/kg bw/day of OAPP (LOAEL) produced distinctly test item-related effects (reduced body weight and enhanced liver weights with hepatocellular vacuolation) in male and female rats (F1-generation). The NOAEL was determined to be 40 mg/kg bw/day, based on inhibited body-weight development.

2.) In the oral short-term repeated dose toxicity study (OECD TG 422, Combined Repeated Dose Toxicity study with Reproduction / Developmental Toxicity Screening Test), repeated oral dietary exposure to approximately 337 mg/kg bw/day of OAPP produced minor toxic effects (reduced body weights, body weight gain, and food consumption) in male and female rats (P0-generation). The NOAEL was determined to be 97 mg/kg bw/day, while the LOAEL was 337 mg/kg bw/day, the highest dose tested.

3.) In the dermal short-term repeated dose toxicity study (OECD TG 410, Repeated Dose Dermal Toxicity: 21/28-Day Study), there was no evidence of toxicologically significant systemic adverse effects that may have been attributed to the test substance at doses of up to 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-01 to 2017-06-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Combined with the Extended One-Generation Reproduction Toxicity Study (EOGRTS), OECD 443: F1-genenration used for Repeated dose study according to OECD 408.
Principles of method if other than guideline:
The study was combined with the Extended One-Generation Reproduction Toxicity Study (EOGRTS), OECD 443: Male and female F1 pups were used for this OECD 408 study: After weaning (on PND 21), animals were treated for 100 days. Recovery animals (Control and High-dose groups) of this F1-generation were derived from Cohort 1B and kept for 30 additional days after the treatment had finished.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH; Batch no. 38900
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
- Expiration date of the lot/batch: 13 February 2018
- Purity test date: 2015-03-24

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, <70 RH%), protected from light
- Stability under test conditions: Not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material): Light yellow to colourless liquid, at low temperature tendency to crystallisation

OTHER SPECIFICS:
- Purity: 100% (UVCB)
- Name of test material (as cited in study report): Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol, OAPP
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI
Details on species / strain selection:
The rat is regarded as suitable species for toxicology and reproduction studies. The Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. The same strain was used in the previous experimental work of the project performed at the Test Facility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
(Further details see IUCLID entry 7.81)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
Animals of F1 generation were group-housed (2 animals of the same sex per cage) after the cohort selection, wherever possible. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no

DETAILS OF FOOD AND WATER QUALITY: tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15.2 – 27.0°C (target range: 22 ± 3°C)
- Humidity (%): 20 – 74% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light (from 6.00 a.m. to 6.00 p.m.)

IN-LIFE DATES: From: 2016-10-27 To: 2017-06-14
Route of administration:
oral: feed
Vehicle:
corn oil
Remarks:
used as the vehicle to facilitate mixing of the test item into the diet to obtain the desired dietary concentration.
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Each diet concentration was prepared as one batch of 100-120 kg/concentration level at the first preparation time, and variable amounts at the further preparation time (30-200 kg/concentration level), depending on the available stability data and/or the expiry date of the diet.
- Mixing appropriate amounts with (Type of food): The test material (OAPP) was incorporated into ssniff® SM R/M-Z+H “Complete Feed for Rats and Mice-maintenance” by ssniff Spezialdiäten GmbH (D-59494 Soest, Germany) to generate the test concentrations required for the study (150, 500 and 1500 ppm). Test material (OAPP) was incorporated into the diet using a solution in corn oil and mixed for up to approximately 12 minutes (approximately 6 minutes for premix preparation, and 6 minutes for preparation of the complete diets). The diets were always produced in an ascending order regarding the test item concentration. Similar diet preparation procedures were used to generate control diet (0 mg OAPP/kg diet).
- Storage temperature of food: Prepared diets were stored at room temperature under dry conditions in sewed bags pending and during transport to CiToxLAB Hungary Ltd. At CiToxLAB Hungary Ltd., the prepared diet bags were stored in areas designated for diet storage at room temperature (approximately 15-21°C), under dry conditions, pending transfer to animal room at approximately 22 ± 3°C for animal feeding.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn Oil (Corn oil was used as the vehicle to facilitate mixing of the test item into the diet to obtain the desired dietary concentration) supplied by Henry Lamotte GmbH (Bremen, Germany)
- Concentration in vehicle: 4%
- Lot/batch no. (if required): Batch Numbers: 16-3329 (8001046002) / 16-3424 (8001213001), 16-3481 (8001409001) / 17-3551 (8002635001); Expiry dates: November 2017 / March 2018 / May /2018 / July 2018)
- Purity: A quality of Ph.Eur.8 (2014) & BP 2016
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the diets for homogeneity and concentration of OAPP was performed at the designated Test Site (Test Site #1) using a validated GC method [6]. Test-item containing diet samples of an appropriate weight were collected from at least five different places of the diet container (in duplicates) from each dose group at least before the start, in the middle and near the end of the use of each batch of diets during the study to determine concentration and homogeneity. At least one sample (in duplicate) was taken from the control diet for concentration analysis at each occasion.
Duration of treatment / exposure:
100 days
Frequency of treatment:
continuous in the diet
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
150 ppm
Remarks:
Low Concentration (150 mg/kg diet), corresponds to ca. 12 mg/kg bw/day (F1 from PND21-PND121, measured)
Dose / conc.:
500 ppm
Remarks:
Intermediate Concentration (500 mg/kg diet nominal) corresponds to ca. 40 mg/kg bw/day (measured, F1 from PND21-PND121)
Dose / conc.:
1 500 ppm
Remarks:
High Concentration (1500 mg/kg diet nominal), corresponds to ca. 122 mg/kg bw/day (measured, F1 from PND21 to PND121)
No. of animals per sex per dose:
- At least 20 animals/sex/concentration of the F1 generation (Cohort 1A) from the OECD 443 study with which the 408 was combined
- 30-day recovery groups (Cohort 1B) - 5 animals/sex in the Control and High-dose group
Control animals:
yes, concurrent vehicle
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels (equivalent to dietary concentrations of 150, 500 and 1500 mg/kg diet) were selected by the Sponsor based on available data and information from previous experimental work, including the available results of a preliminary Dose Range Finding (DRF) palatability study (Study code: 15/094-220PE) and an OECD No. 422 study ((Study code: 15/094-220P) performed at the Test Facility.

In the OECD No. 422 study, OAPP was administered in diet at the concentrations of 300, 1250 and 5000 ppm, (equivalent to dietary doses of approximately 24.5, 97.1 and 337.6 mg/kg bw/day in the Low-, Mid- and High-dose groups, respectively) to Wistar rats for at least 28 days. Administration of OAPP caused significantly reduced body weights, reduced body-weight gain and reduced food consumption in the High-dose of both sexes; slight differences were seen in the Mid-dose females.

Organ weight and macroscopic observations at necropsy showed hepatic enlargement in both sexes at the High-dose and in Mid-dose males. At histopathology evaluation, hepatocellular vacuolation was also detected confirming the macroscopic observations. However, in the absence of effects on the liver function (as indicated by the clinical chemistry parameters) the hepatic changes were considered as probably an adaptive rather than an adverse effect. Based on this it was decided to add 30 day recovery groups to the OECD 408 study, to confirm this is an adaptive response. The No-Adverse-Effect Level (NOAEL) of OAPP for systemic, reproductive and developmental toxicity was concluded to be1250 ppm (97.1 mg/kg bw/day).

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Adult animals inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day as practical.

DETAILED CLINICAL OBSERVATIONS: Yes (see Report, Appendix 4.1.3)
- Time schedule: Detailed clinical observations were made on all adult animals at least prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter. These observations were made outside the home cage in a standard arena, at similar times, generally during the morning. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards). Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes (see Report, Table 20, and Appendix 4.2.3)
- Time schedule for examinations: Body weight was recorded with a precision of 1 g at weaning (PND 21, prior to the start of treatment which was designated as Day 1 of individual treatment), then at least weekly, including on Day 100 (last treatment day) and prior to necropsy (if scheduled necropsy, fasted, on Day 101).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g twice per week. Weekly food consumption was calculated for reporting purposes. Determination of food consumption of Cohort 1A of F1 generation was performed twice per week. The first measurement of given food was made at weaning (PND 21, i.e. Day 1). The remaining, non-consumed food was weighed twice per week with a precision of 1 g. Food hoppers were replenished more frequently if required.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examination was conducted in all animals of Cohort 1A soon after the start of treatment (Week 6) and in the Control (Group 5) and High-dose (Group 8) animals, during Week 12/13 of the treatment. As no treatment-related alterations were found, no additional animals of Low- (Group 6) and Mid- (Group 7) dose groups or recovery animals were examined near/at termination.
- Dose groups that were examined:all animals of Cohort 1A. All animals of Control (Group 5) and High-dose (Group 8) Groups.

HAEMATOLOGY: Yes (Report, Table 24, and Appendix 4.4.3)
- Time schedule for collection of blood: Prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (pentobarbital anaesthesia) - After an overnight period of food deprivation of animals (fasting), blood samples were collected by heart puncture under pentobarbital anaesthesia, for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (in tubes with sodium citrate as anticoagulant)
- Animals fasted: Yes (overnight)
- How many animals: all surviving animals

CLINICAL CHEMISTRY: Yes (Report, Table 25, and Appendix 4.4.3)
- Time schedule for collection of blood: Prior to scheduled necropsy
- Animals fasted: Yes (overnight)
- How many animals: all surviving animals

URINALYSIS: Yes (Report, Appendix 4.4.3)
- Time schedule for collection of urine: Prior to scheduled necropsy (collected overnight (for approximately 16 hours))
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (with no access to food, but with access to water)

NEUROBEHAVIOURAL EXAMINATION: Yes (Report, Table 22, and Appendix 4.1.5)
- Time schedule for examinations: Towards the end of the treatment period, during Week 12/13, but not earlier than in Week 11
- Dose groups that were examined: each animal of Cohort 1A which was used to populate the OECD 408 study
- Battery of functions tested: sensory activity / grip strength / motor activity / other: functional observation battery, including measurements of the landing foot splay and fore/hind limb grip strength; and motor activity assessment.

IMMUNOLOGY: For the investigation of possible pre- and post-natally induced immunotoxic effects, Cohort 1A animals were subjected to extra steps at termination including weighing of extra organs/tissues associated with the immune system and splenic lymphocyte subpopulation analysis.

THYOID HORMONE ANALYSIS: Blood sampling was done for thyroid hormone analysis (TSH, T4 and/or T3) of Cohort 1A animals (see Report, Table 26
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy (scheduled on PND111) for Cohort 1A animals was conducted on PND111 (males / females) (09-15 June 2017)
Necropsy for recovery animals (Cohort 1B) was conducted on PND141 (males / females) (10 July 2017)

HISTOPATHOLOGY: Yes (see Report, Appendix 1 (Pathology Report)

ORGAN WEIGHTS: Yes (Report, Table 26/27)
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 (when using Provantis).
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of continuous numeric data:
The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.
 
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item treatment-related clinical signs were detected for any animals of cohort 1A (used for the 90-day repeated dose toxicity study). Minor clinical signs were recorded for some animals as follows. Red discharge was noted for one Control male (near the snout in the period of PND 98-119) and one Mid-dose male (near the left eye in the period of PND 84-119). Hyperactivity was recorded for a Low-dose female in the periods of PND 29-121.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no statistically significant, test item-related effect on the Low-dose F1A-animals. There was a slightly significant decrease (about 7%, p<0.05) in the body weight of the High-dose F1A-males (at PND120), while the terminal body-weight gains (PND21-120) remained insignificant for all male groups. On the other hand, the terminal mean body weights of females of the Mid- and High-dose group were statistically lower than those of the controls (-9 and -18 %, respectively), more marked for the mean body-weight gains from PND21 to PND120 (-10% and -20%, respectively) (see Report Table 20). The significant decreases in mean body weight in the male and female High-dose groups are considered to be treatment-related.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test item-related reduced food consumption was observed for High-dose females of Cohort 1A (minus 10.6% after PND120, p<0.01), but no similar effect was observed in High-dose males, or in the Mid- and Low-dose groups of either sex (see Report, Table 21).

Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test item-related changes were noted at ophthalmoscopy examination. All examined animals were found to be normal even at early time-point (PND 36-42) as well as before termination (PND 101-110).
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared to the controls, there were no differences in any haematological parameters that could be considered toxicologically significant in any test item-treated group of Cohort-1A males and females, although statistically significant in some cases: decreased mean cell haemoglobin and MCC (p<0.05 or p<0.01) in the Mid- and High-dose males, increased platelet count (p<0.05) in High-dose females, and decreased relative amount of reticulocytes in the Mid- and High-dose females (p<0.05 and p<0.01, respectively) (see Report, Table 24, and Appendix 4.4.3). However, all those values did not suggest a dose response, while the same time were within the normal physiological and historical control range.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes on the serum chemistry parameters of the animals in Cohort 1A, although certain parameters showed statistically significant differences when compared to the controls: decreased cholesterol concentration in the Mid- and High-dose males (p<0.01), decreased albumin concentration and albumin/globulin ratio in High-dose females (p<0.05 or <0.01, respectively) (see Report, Table 25, and Appendix 4.4.3).

Urinalysis findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in the test item-exposed groups were observed in the urinalysis of Cohort 1A, concerning urine volume, pH or specific gravity, urinary constituents and appearance (see Report, Appendix 4.4.3).

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test item-related effects in the neurological assessment, as there were no observed differences in animal behaviour, general physical condition (grip strength, locomotor activity) or in the reactions to different types of stimuli in the control or test item-treated groups (see Report, Table 22, and Appendix 4.1.5).

Immunological findings:
no effects observed
Description (incidence and severity):
In an immunophenotyping analysis on splenic lymphocyte samples of Cohort 1A to determine the relative and absolute counts of six different lymphocyte subpopulations, there were no test item-related shifts in the immunological steady-state distribution of "helper" (CD4+) or cytotoxic (CD8+) thymus-derived lymphocytes or natural killer (NK) cells in males or females (see Report, Table 30/31, and Appendix 10).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increases, considered test item-related, were present in the liver weights in both sexes of the High-dose group and in the Mid-dose males (see Report, Table 27/28). These differences were supported by test item-related histopathological findings.
Other statistical differences were found in High-dose males: brain, kidney and thyroid (only when adjusted to body weight), and in High-dose females: brain, adrenals, thyroid, uterus (adjusted to body weight), heart, kidney, spleen and ovary. But based on microscopic examination these organ-weight differences were considered as secondary changes due to the body weight effect, without evidence of a direct effect of the test item. For further information: see `Details on results´.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A:
Treatment-related changes were recorded in the liver of High-dose males. No treatment-related changes were noted in the High-dose females. Pale mottled/diffuse discoloration was seen in 4/22 males of the High-dose only (a similar incidence of this finding as in the parental (F0) generation animals). All other changes were incidental, a common background or related to the oestrous cycle.

Cohort 1B:
At the terminal necropsy, a total of 2/18 High-dose males had hepatic changes, pale and enlarged liver in 1/18 and pale liver in 1/18 males with liver weights were slightly increased in the High-dose only. All other changes were considered to be incidental or common background findings. In recovery subset animals, no significant liver-weight or any hepatic effects and no other no treatment-related changes were detected at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A:
Treatment-related histopathological findings were noted in examined High-dose animals.
There was a mixed form of hepatocellular vacuolation observed in the livers of High-dose males and females: centrilobular (mainly) and periportal zonation were seen. The severity varied from minimal to moderate with frequency of 19/22 males and 13/24 females. A severe intensity with diffuse distribution altered liver was found in 1/22 males. No similar vacuolation was recorded in controls. This observation was considered to be non-adverse. In addition to the standard histopathology examination, for F1 parental females only, a quantitative assessment of primordial follicles and corpora lutea was conducted. There were no meaningful differences in total number (left and right ovaries) of primordial follicles and corpora lutea between Control and High-Dose females

Cohort 1B:
High-dose recovery animals were examined (The 1A cohort provided the histopathology evaluation for the F1 animals). No treatment-related microscopic changes were observed in examined males and females.
Hepatocellular vacuolation of the liver was observed in 2/5 males exposed at a dietary concentration of 1500 mg/kg diet; however, macrovesicular forms were considered to be common background observations similar to those commonly seen in control animals. Hence, it was considered there was no effect in these recovery animals.
In addition, a quantitative assessment of primordial follicles and corpora lutea was conducted in the females according to the Study plan. There were no meaningful differences in total number (left and right ovaries) of primordial follicles and corpora lutea between Control and High-dose females.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Cohort 1A
Additional parameters (balanopreputial separation for males, vaginal opening and oestrus cycle for females) were also examined to follow the sexual development of Cohort 1A animals.
Balanopreputial separation: A slightly slower but not statistically significant development in the High-dose males was observed in the Cohort 1A (retardation of about 1-2 days (PND42 vs. PND40 to 41) (see Report, Figure 15, and Appendix 3.9). However, since the body weight at the time of balanopreputial separation was significantly lower than controls, the slower development was considered to be secondary to lower body weight and not a direct test item effect.

Vaginal patency: Vaginal patency (vaginal opening) was normal for all female pups beginning on PND 22. The body weight of each female was recorded on the day of vaginal patency. No effect on this parameter was observed in the Low-, Mid- and High-dose groups of Cohort 1A females (see Report, Figure 16, Table 23, and Appendix 3.9).

Oestrus cycle: The Oestrous cycles did not show significant differences between the groups.

Sperm Analysis: Sperm motility and morphology as well as number of sperms were examined in the study for Cohort 1A. No treatment-related effect was seen in any dose groups of this cohort (see Report, Table 29, and Appendix 4.6.3).

Thyroid hormone analysis: No toxicologically relevant changes in the test item exposed groups were observed in the thyroid hormone values of Cohort 1A (see Report, Table 26, and Appendix 4.4.3).
A statistically higher value (p<0.05) was recorded for the High-dose females for T4 (of 45.45 pg/mL), however the Parental T4 range (47.9 - 60.8 pg/mL for animals of approximately the same age) shows that the control mean for the 1A group is relatively low. The High-dose female 1A data is not higher than the normal range, hence the statistical difference is not considered to be of biological significance.

Cohort 1B
Cohort 1B animals were dedicated to obtaining additional histopathology data in cases of suspected reproductive or endocrine toxicants, or when results from Cohort 1A were equivocal. No such an assessment was deemed necessary in the study. Hence, the data from in-life and at clinical pathology and necropsy were tabulated and included in the Appendices attached to the study report but are not reported, since the requirement for this additional data was not triggered. The macroscopic data from necropsy has been provided to supplement the findings in the 1A cohort. Additionally, the histopathology data of the recovery animals of the 1B cohort has also been provided.

The following information for Cohort 1B was collected and/or parameters measured during the in life phase: clinical observations, body weight and body weight gain, food consumption, clinical pathology (haematology, clinical chemistry, urinary analysis, necropsy findings, organ weights (absolute and adjusted to body / brain weight) and histopathology. All the results are provided in the appropriate appendices attached to the study report, but no formal evaluations were made, as it was not necessary based on the Cohort 1A results.
Details on results:
COHORT 1A:
CLINICAL CHEMISTRY:
There were no test item-related changes on the serum chemistry parameters of the animals in Cohort 1A, although certain parameters showed statistically significant differences when compared to the controls: decreased cholesterol concentration in the Mid- and High-dose males (p<0.01), decreased albumin concentration and albumin/globulin ratio in High-dose females (p<0.05 or <0.01, respectively).

Cholesterol: A similar decrease but without statistical significance was seen in the High-dose females (see Report, Table 25, and Appendix 4.4.3). However, the observed mean values were within the normal physiological range (mean of historical control ± 2-times standard deviation, SD) in all cases (males and females). Furthermore, the control mean was at the maximum of the historical range, while the High-dose was in the middle of the historical range. Hence, from this data alone, it would have been considered to be unrelated to treatment. In this study in the P0 generation, the control values were also relatively high, and there were slightly but statistically significantly (p<0.05) lower cholesterol values, hence a relationship with treatment cannot be totally excluded, but the High-dose group values were in the middle of the historical normal range. The differences in the High-dose males in 1A-cohort are considered to be of equivocal relationship with treatment, but in the absence of any other associated findings, when the values were considered completely normal, were considered not to reflect an adverse change .

Protein: Decreased values for parameters related to protein content (total protein concentration, albumin concentration and albumin/globulin ratio) were observed in High-dose females, the difference from control was statistically significant for albumin and A/G ratio (p<0.05 or <0.01, respectively). However, no similar trends were seen in High-dose males (actually in High-dose males an opposite change, statistically significant increase (p<0.05) in total protein content was detected). Furthermore, all the observed values were within the normal physiological range (mean of historical control ± 2SD). Therefore, the observed differences between values were considered as having no biological relevance (the actual values of female control animals were higher than usual based on the historical control database) and not being related to test item administration.

Ions: Statistically significant changes (at p<0.05 level) were recorded for the concentrations of some ions: increased sodium and calcium concentration were noted for High-dose males, and increased chloride concentration was noted in High-dose females when compared to control animals. However, the differences were minor (less than 5%), no similar trends were seen in the other sex in any of those cases. Furthermore, all those data were within the normal physiological control and/or historical control range. Therefore, they were considered as animal variability, not related to the test item.


BODY WEIGHT AND WEIGHT CHANGES:
There was no test item related effect on the Low dose group animals. There was no effect on body weight of males at any dose level. Females in the Mid- and High-dose groups had a lower starting body weight at the High dose (and to some extent at the Mid dose) and they had a lower growth rate compared to controls at the Mid and High dose level.

Although statistically significant decrease (by 7%, p<0.05) was observed in the body weight at the end of the evaluation period (PND 120) for High-dose males when compared to the control, this was not considered as clear evidence of test item effect, as the starting body weight at weaning (on PND 21) also showed a similar difference (approx. 8%, p<0.05). As no significant difference was seen in the body weight gain of the High-dose males during the PND 21-120 period, the observed values were considered as showing no test item-related effect. Neither were there significant changes of body weight in the Mid- and Low-dose groups.

However, in case of Cohort 1A females, the original difference in the starting body weight of Mid- and High dose animals compared to control (by 4.9% and 9.0%) became wider by the end of the evaluation period (9.0% in case of Mid-dose females and 18.1% in case of High dose females, both significant at p<0.01). Furthermore, the body weight gain during this period (PND 21-120) was statistically significant in the Mid- and High-dose females (10.3% and 20.3%, p<0.01 in both cases) compared to the control group. These values indicated a test item-related effect in the females of the Mid-and High-dose group, although the impact of a lower starting body weight is difficult to evaluate. There were no significant changes in the Low dose females of this cohort.

ORGAN WEIGHT:
Terminal body weights of males and females were statistically significantly lower in the terminal body weights (by 9.0%, p<0.01) was also observed in Mid-dose females. No changes in terminal body weights were observed in Mid-dose males or Low-dose animals when compared to the controls.

Statistically significant differences, considered test item-related, were present in the liver weights in both sexes of the High-dose group. These differences were supported by test item-related histopathological findings. There was an increase in absolute and relative (to body and brain) liver weights in the High-dose males, and for body weight-adjusted liver weights in the High-dose females. In the High-dose males, absolute liver weights were statistically significantly higher by 20.1% (p<0.01), body-related by 29.6% (p < 0.01), and by 19.9% (p < 0.01) when brain-related. In High-dose females, liver weight-adjusted body were increased by 15.0% (p < 0.01) but values were slightly lower than control for absolute and brain-adjusted weights. A statistically significant increase considered test item-related was defined in the liver of Mid-dose males. When related to body and to brain weights, liver weights were larger by 11.5% (p<0.01) and by 10.8% (p <0.05), respectively. No statistically significant differences were reported in Mid-dose female livers.

In both sexes of the High-dose group, the treatment-related lower body weight had a consequence with some statistically significant lower organ weights. But based on microscopic examination these organ weight differences were considered as secondary changes due to the body-weight effect, without evidence of a direct effect of the test item. These statistical differences were seen in High-dose males concerning brain, kidney and thyroid (only when adjusted to body weight), and in High dose females concerning brain, adrenals, thyroid, uterus (adjusted to body weight), heart, kidney, spleen and ovary.

There were no significant differences considered to be treatment-related in the Low-dose group (males and females) compared to controls.

GROSS PATHOLOGY:
Treatment-related changes were recorded in the liver of High dose males. No treatment-related changes were noted in the High dose females.

Pale mottled/diffuse discoloration was seen in 4/22 males of the High-dose only (a similar incidence of this finding as in the parental (F0) generation animals). All other changes were incidental, a common background or related to the oestrous cycle.

HISTOPATHOLOGY: see Results of Examination

Key result
Dose descriptor:
NOAEL
Effect level:
42 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no

Table 7. Selected body weight parameters of Cohort 1A animals

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated males

20

23

21

22

 

Male, Body weight on PND21 (g)

68.7

67.5

64.9

63.1**

D

difference (%)

-1.8

-5.5

-8.2

 

Male, Body weight on PND120 (g)

595.3

610.3

584.6

555.3*

DN

difference (%)

2.5

-1.8

-6.7

 

Male, Body weight gain PND21-120 (g)

526.6

542.8

519.7

492.2

NS

difference (%)

3.1

-1.3

-6.5

 

Number of evaluated females

22

23

21

24

 

Female, Body weight on PND21 (g)

65.8

62.9

62.6

59.9**

D

difference (%)

-4.5

-4.9

-9.0

 

Female, Body weight on PND120 (g)

331.3

312.9

301.5**

271.4**

DN

difference (%)

-5.6

-9.0

-18.1

 

Female, Body weight gain PND21-120 (g)

265.5

250.0

238.9**

211.5**

DN

difference (%)

-5.8

-10.0

-20.3

 

Notes: Data (group mean values) were rounded to one decimal place.

PND = post-natal day

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

D: Dunn test; DN: Dunnett’s Multiple Range Test, NS: Statistically not significant when comparedto control

Table 8. Selected food consumption parameters of Cohort 1A

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated males

20

24

21

22

 

Male, Food consumption (PND21-120) (g/day)

26.88

28.64

27.78

26.78

NS

difference (%)

6.6

3.4

-0.4

 

Number of evaluated females

22

23

21

24

 

Female, Food consumption (PND21-120) (g/day)

19.43

19.40

19.01

17.37**

DU

difference (%)

-0.2

-2.2

-10.6

 

Notes: Data (group mean values of daily food consumption) were rounded to two decimal places.

PND = post-natal day

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DU: Dunn test, NS: Statistically not significant compared to control

Table 9. Selected FOB and SMART parameters of Cohort 1A

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated male animals

20

23

21

22

 

Males, Landing foot splay (hind paws)

90.0

78.23

83.70

76.41

NS

Males, Grip-strength (forelimbs)

1809.2

1813.3

1783.85

1768.9

NS

Males, Grip-strength (hind limbs)

453.3

477.4

436.05

454.1

NS

Males, Total travelled distance (cm)

7992.6

8767.8

8070.167

8632.9

NS

Number of evaluated female animals

22

23

21

23

 

Females, Landing foot splay (hind paws)

75.5

65.7

67.2

60.2*

DN

Females, Grip-strength (forelimbs)

1321.0

1269.5

1274.5

1275.0

NS

Females, Grip-strength (hind limbs)

344.0

318.1

329.2

294.3

NS

Females, Total travelled distance (cm)

9523.8

11426.3

10894.8

10352.0

NS

Notes: Data (group mean values) are rounded to one decimal place.

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DU: Dunn Test, DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

Table 10. Summary of selected haematology parameters (Cohort 1A parental animals)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, Mean Cell Haemoglobin (pg)

18.06

17.35*

17.30*

17.22**

DU

difference (%)

-3.9

-4.2

-4.6

 

Male, MCHC (g/dL)

33.88

33.05

32.76**

32.63**

DU

difference (%)

-2.4

-3.3

-3.7

 

Male, Platelet count (K/μL)

965.8

995.6

990.5

984.8

NS

difference (%)

3.1

2.6

2.0

 

Male, Relative amount of reticulocytes (%)

2.420

2.409

2.471

2.382

NS

difference (%)

-0.5

2.1

-1.6

 

Female, Mean Cell Haemoglobin (pg)

18.63

18.49

18.40

18.10

NS

difference (%)

-0.8

-1.3

-2.9

 

Female, MCHC (g/dL)

33.27

33.58

33.15

33.08

NS

difference (%)

0.9

-0.4

-0.6

 

Female, Platelet count (K/μL)

862.6

895.5

965.2

959.0*

DN

difference (%)

3.8

11.9

11.2

 

Female, Relative amount of reticulocytes (%)

2.60

2.42

2.30*

2.033**

DU

difference (%)

-7.0

-11.4

-21.8

 

Notes: Data (group mean values, n=20-24) were rounded as appropriate (to 1-3 decimal places).

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DU: Dunn Test, DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

Table 11. Selected clinical chemistry parameters of Cohort 1A animals

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, Cholesterol (mmol/L)

2.190

1.930

1.758**

1.669**

DN

difference (%)

-11.8

-19.7

-23.8

 

Male, Total protein (g/L)

57.92

58.19

58.90

60.18*

DN

difference (%)

0.5

1.7

3.9

 

Male, Albumin (g/L)

31.59

31.77

32.12

33.01

NS

difference (%)

0.6

1.7

4.5

 

Male, A/G ratio

1.20

1.20

1.20

1.22

NS

difference (%)

-0.4

0.4

1.9

 

Male, ALKP (U/L)

102.7

92.8

90.5

105.6

NS

difference (%)

-9.6

-11.8

2.9

 

Male, ALT / GPT (U/L)

43.5

47.9

52.8

52.9*

DU

difference (%)

10.0

21.3

21.6

 

Male, Bile acid (μmol/L)

12.770

12.867

14.974

16.295*

DU

difference (%)

0.8

17.3

27.6

 

Female, Cholesterol (mmol/L)

2.408

2.117

2.111

1.943

NS

difference (%)

-12.1

-12.3

-19.3

 

Female, Total protein (g/L)

67.05

64.49

65.93

64.51

NS

difference (%)

-3.8

-1.7

-3.8

 

Female, Albumin (g/L)

41.50

39.35

40.05

37.99*

DN

difference (%)

-5.2

-3.5

-8.5

 

Female, A/G ratio

1.636

1.565

1.548

1.442**

DN

difference (%)

-4.3

-5.4

-11.9

 

Female, ALKP (U/L)

52.9

50.6

62.6

73.4**

DU

difference (%)

-4.3

18.4

38.7

 

Female, ALT / GPT (U/L)

46.9

52.6

55.5

50.8

NS

difference (%)

12.2

18.5

8.3

 

Female, Bile acid (μmol/L)

14.050

16.408

16.636

17.875*

DU

difference (%)

16.8

18.4

27.2

 

Notes: Data (group mean values, n=20-22) were rounded as appropriate (to 1-3 decimal places).

ALT (GPR): Alanine Aminotransferase (Glutamate-pyruvate transaminase); ALKP: Alkaline phosphatase

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test; DU: Dunn test; NS: Statistically not significant when compared to control

Table 12. Organ weight data of Cohort 1A males

 

Organ weight

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated animals

20

23

21

22

 

Terminal body weight, g

571.2

584.9

560.1

530.1*

DN

(difference %)

2.4

-1.9

-7.2

 

Liver (absolute), g

15.824

16.906

17.300

19.003**

DN

(difference %)

6.8

9.3

20.1

 

Liver (relative to body), %

2.770

2.889

3.089**

3.589**

DU

(difference %)

4.3

11.5

29.6

 

Liver (relative to brain), %

692.08

739.60

767.05*

829.84**

DN

(difference %)

6.9

10.8

19.9

 

Notes: Organ weight data (group mean values) were rounded to two or three decimal places.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test, DU: Dunn’s test, NS: Statistically not significant compared to control

 

Table 13. Organ weight data of Cohort1 1A females

 

Organ weight

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated animals

22

23

21

22

 

Terminal body weight, g

317.1

298.4

288.6**

257.7**

DN

(difference %)

-5.9

-9.0

-18.8

 

Liver (absolute), g

10.223

9.906

9.680

9.570

NS

(difference %)

-3.1

-5.3

-6.4

 

Liver (relative to body), %

3.234

3.328

3.365

3.717**

DU

(difference %)

2.9

4.1

15.0

 

Liver (relative to brain), %

497.73

481.71

473.12

471.77

NS

(difference %)

-3.2

-4.9

-5.2

 

Notes: Organ weight data (group mean values) were rounded to two or three decimal places.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test, DU: Dunn’s test, NS: Statistically not significant compared to control

Table 14. Immunophenotyping of Cohort 1A (absolute counts)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

 

Number of analysed samples

20

21

21

22

NS

Number of splenocytes (cell/μL)

206.9

178.6

241.0

240.9

NS

Number of B cells (cell/μL)

84.2

75.5

103.8

98.6

NS

Number of NK cells (cell/μL)

8.6

7.6

7.5

10.5

NS

Number of NKT cells (cell/μL)

11.0

9.1

13.8

11.0

NS

Number of T cells (cell/μL)

79.8

64.6

85.0

86.4

NS

Number of Helper T cells (cell/μL)

54.4

42.5

61.0

56.2

NS

Number of Cytotoxic T cells (cell/μL)

18.8

14.8

20.0

21.3

NS

Females

 

Number of analysed samples

22

21

21

24

NS

Number of splenocytes (cell/μL)

203.9

176.7

228.7

237.8

NS

Number of B cells (cell/μL)

83.6

77.0

96.2

102.5

NS

Number of NK cells (cell/μL)

8.4

6.5

8.0

8.5

NS

Number of NKT cells (cell/μL)

10.5

8.5

13.3

9.6

NS

Number of T cells (cell/μL)

75.0

66.2

83.9

87.1

NS

Number of Helper T cells (cell/μL)

50.6

45.5

61.1

59.1

NS

Number of Cytotoxic T cells (cell/μL)

17.3

14.6

18.5

20.1

NS

Notes: Data (group mean values) are rounded to one decimal place.

NS: statistically not significant, according to the decision tree (relevant phase report), where Dunnett’s test is used for significance.

 

Table 15. Immunophenotyping of Cohort 1A (relative counts)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

 

Number of analysed samples

20

21

22

22

NS

Number of B cells (cell/μL)

40.5

41.1

42.6

40.2

NS

Number of NK cells (cell/μL)

4.2

4.4

3.3

5.3

NS

Number of NKT cells (cell/μL)

5.4

5.2

6.6

4.9

NS

Number of T cells (cell/μL)

39.0

36.9

35.5

36.5

NS

Number of Helper T cells (cell/μL)

68.2

66.2

91.8

65.4

NS

Number of Cytotoxic T cells (cell/μL)

22.9

22.4

32.9

23.9

NS

Females

 

Number of analysed samples

22

21

21

24

NS

Number of B cells (cell/μL)

40.2

42.7

41.6

42.8

NS

Number of NK cells (cell/μL)

4.3

3.7

3.7

3.8

NS

Number of NKT cells (cell/μL)

5.2

4.9

6.5

4.2

KW*

Number of T cells (cell/μL)

37.5

38.2

36.6

36.9

NS

Number of Helper T cells (cell/μL)

66.8

68.3

89.7

67.7

NS

Number of Cytotoxic T cells (cell/μL)

23.2

22.3

27.5

23.2

NS

Notes: Data (group mean values) are rounded to one decimal place.

NS: statistically not significant, according to the decision tree (relevant phase report), Dunnett’s test for significance.

Table 16. Summary of thyroid hormone analysis (Cohort 1A)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, T4 (ng/mL)

56.25

51.91

51.97

56.20

NS

difference (%)

-7.7

-7.6

-0.1

 

Male, TSH (pg/mL)

1847.8

1689.7

2169.1

2414.1

NS

difference (%)

-8.6

17.4

30.6

 

Male, Terminal body weight (g)

571.2

584.9

560.1

530.1*

DN

difference (%)

2.4

-1.9

-7.2

 

Male, Thyroid & parathyroid weight (g)

0.0322

0.0345

0.0325

0.0347

NS

difference (%)

7.5

1.2

7.9

 

Female, T4 (ng/mL)

33.97

38.89

33.45

45.45*

DN

difference (%)

14.5

-1.5

33.8

 

Female, TSH (pg/mL)

2118.7

1505.0

1441.9

2606.9

NS

difference (%)

-29.0

-31.9

23.0

 

Female, Terminal body weight (g)

317.1

298.4

288.6**

257.7**

DN

difference (%)

-5.9

-9.0

-18.8

 

Female, Thyroid & parathyroid weight (g)

0.0250

0.0259

0.0235

0.0242

NS

difference (%)

3.6

-6.1

-3.1

 

Notes: Data (group mean values, n=10) were rounded to one to four decimal places

Conclusions:
Based on the effects observed in Cohort 1A and Cohort 1B of this study, the NOAEL for systemic toxicity was determined to be 42 mg/kg bw/day, corresponding to 500 mg OAPP/kg diet (nominal). Additionally, exposure to the test material did not result in any immunotoxic effects. The effect was more marked in the treated females.
Executive summary:

A key combined extended one-generation reproductive toxicity (EOGRTS) / sub-chronic oral toxicity study was conducted according to Guideline recommendations (OECD 443 / OECD 408) to evaluate specific life stages not covered by other types of toxicity studies and to test for effects that may occur as a result of pre- and post-natal chemical exposure to the test material (Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (OAPP), EC# 700 -960 -7). The OECD 443 study was combined with the OECD 408 (90-day rat) study using the F1 generation (hence exposure was in utero to weaning, then effectively further 100 days), to evaluate sub-chronic toxicity of the test material.

 

The test material was administered continuously in the diet to selected pups (male and female Wistar rats) of the F1 generation (Cohort 1A: at least 20 animals/sex/group) at nominal concentrations in the diet of 0, 150, 500, or 1500 ppm [mg/kg diet] for 100 days after weaning, i.e. from PND21 through PND120. This part of the study also included 30-day recovery animals (Cohort 1B: 5 males and 5 females in the Control and High-dose groups, respectively).

 

There were no mortalities after weaning, no adverse clinical signs, and no effects were observed on the neurotoxicity parameters evaluated.

 There was no statistically significant, test item-related effect on the Low-dose F1A-animals. There was a slightly significant decrease (about 7%, p<0.05) in the body weight of the High-dose F1A-males (at PND120), while the terminal body-weight gains (PND21-120) remained insignificant for all male groups. On the other hand, the terminal mean body weights of females of the Mid- and High-dose group were statistically lower than those of the controls (-9 and -18 %, respectively), more marked for the mean body-weight gains (-10% and -20%, respectively (from PND21-120) (see Report Table 20). The decreases in mean body weight in the male and female High-dose groups are considered to be treatment-related.

 

No adverse treatment-related effects on haematology, clinical chemistry, or urine analysis were observed in any dose group. All examined animals were found to be normal during the ophthalmoscopy examinations.

 

Reductions in some organ weights were observed, but considered to be a consequence of lower body weights, and not an adverse effect.

A treatment-related mixed form of hepatocellular vacuolation occurred in the High-dose males and females, visualised as pale lobes seen in some males at necropsy. A statistically significant liver-weight increase, considered treatment-related, was present in both sexes in the High-dose, and to a slight extent in Mid-dose males. Centrilobular (mainly) and periportal zonation were seen at histopathology; similar to the parental animals, the severity varied mainly from minimal to moderate, and there were proportional frequencies viewed in the male and females. The hepatic morphological changes were considered to be an adaptive, non-adverse response of the liver.

 

In the recovery animals (high-dose vs. control groups), there were no significant differences in liver weights and no treatment-related macroscopic or microscopic hepatic changes in examined males and females after the recovery period. This confirms the adaptive, non-adverse nature of the terminal hepatic observations. Additionally, a quantitative assessment of primordial follicles and corpora lutea conducted in terminal and recovery females did not show any meaningful differences in total number of primordial follicles and corpora lutea between Control and High-dose females.

 

Based on the effects observed in Cohort 1A and Cohort 1B of this study, the NOAEL for systemic toxicity was determined to be 42 mg/kg bw/d, administered to rats in the food at a level of 500 mg/kg diet (nominal). Additionally, exposure to the test material did not result in any immunotoxic effects. 

Endpoint:
repeated dose toxicity: oral, other
Remarks:
repeated exposure for 28 and 40 days in OECD 422 study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09-22 - 2015-11-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Final draft report - results reviewed
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (OAPP)
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH, batch No. 38900
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
- Stability under test conditions: found to be stable for 8 days (no longer observation)
- Storage condition of test material: room temperature, exclusion of light
- Expiry date: 2017-02-13
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, D-97633, Sulzfeld, from SPF colony
- Age at study initiation: 12 weeks old at start of mating
- Weight at study initiation: 195 - 250 g (females); 341 - 408 (males), both at onset of the treatment (14 days prior to mating)
- Fasting period before study: no
- Housing: Type II and/or III polycarbonate cages; two animals of the same sex and dose group/cage, but individually during mating and the gestation and lactation period.
- Diet: ad libitum until day 4 post-partum (necropsy on day 5 post-partum)
- Water: ad libitum
- Acclimation period: 5 days (until control diet administration); 12 days (until the start of the treatment).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 25.0°C
- Humidity (%): 40 - 77%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: feed
Vehicle:
corn oil
Remarks:
used as carrier for mixing with the diet
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): once
- Mixing appropriate amounts with (Type of food): ssniff® SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Storage temperature of food: at room temperature, dry conditions

VEHICLE
- Justification for use and choice of vehicle: The test item was incorporated into the diet and mixed for up to approximately 14 min (approx. 6 min for premix preparation, and 4-8 min for preparation of the complete diets) using a solution of test item in corn oil.
- Concentration in vehicle: depending on dose group
- Amount of vehicle: 4% in the diet
Analytical verification of doses or concentrations:
yes
Remarks:
3x in duplicate from five different places of the diet container from each dose group
Details on analytical verification of doses or concentrations:
Validated GC method: During the non-GLP Dose Range Finding study (study code: 15/094-220PE), the stability in diet was demonstrated. During this main study the stability of test item in the diet was confirmed.
Duration of treatment / exposure:
Males: at least 28 days (pre-mating 14 days and mating/post-mating at least 14 days)
Females: at least 42 days (pre-mating 14 days, mating up to 14 days, gestation 22-24 days, and lactation 4 days)
Frequency of treatment:
continuous
Dose / conc.:
300 other: ppm (nominal) (corresponds to 24.5 mg/kg bw/day actual dose received)
Remarks:
Low dose - estimated target ~15 mg/kg bw/d (via diet);
actual dose: 24.5 mg/kg bw/day (calculated) (see Report Tab. 8)
Dose / conc.:
1 250 other: ppm (nominal) (corresponds to 97 mg/kg bw/day actual dose received)
Remarks:
Mid dose - estimated target ~62.5 mg/kg bw/d (via diet)
actual dose: 97.1 mg/kg bw/day (calculated) (see Report Tab. 8)
Dose / conc.:
5 000 other: ppm (nominal) (corresponds to 337 mg/kg bw/day actual dose received)
Remarks:
High dose - estimated target ~250 mg/kg bw/d (via diet);
actual dose: 337.6 mg/kg bw/day (calculated) (see Report Tab. 8)
Dose / conc.:
283 other: ppm (analytical)
Remarks:
Low concentration in diet - analytical (see Report Tab. 7)
Dose / conc.:
1 065 other: ppm (analytical)
Remarks:
Mid concentration in diet - analytical (see Report Tab. 7)
Dose / conc.:
5 235 other: ppm (analytical)
Remarks:
High concentration in diet - analytical (see Report Tab. 7)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A dose-selection and palatability study had been conducted. The palatability of the rodent diet containing OAPP was investigated at 100, 300, 1000, 3000, 10000 and 20000 ppm (mg/kg diet, acc. to study report) for 14 consecutive days (CiToxLAB Study code 15/094-220PE). The low food intake at 10000 and 20000 ppm indicated that these dose levels were above the Maximum Tolerated Dose for an OECD No. 422 study.
- Rationale for animal assignment (if not random): An equal number of animals from each weight group was randomly allocated to each dose group as to ensure that the mean group weights were similar.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 1x/d
- Cage side observations checked in Appendix 1.1.1 and 1.1.2 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to treatment, then 1x/wk

BODY WEIGHT: Yes (see Appendix 1.2.1)
- Time schedule for examinations: All animals on day 0, then 2x/wk, P-females on GD 0, 3, 7, 10, 14, 17 and 20 and on postpartal days 0, 2, and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (see Appendix 1.3)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice (males on day 29, females on PND 5)
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: 4 m/ 5 f
- Parameters checked in Appendix 1.4 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals: 4 m/ 5 f
- Parameters checked in Appendix 1.5 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at sacrifice (males on day 29, females on PND 5)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (see report 4.6.3)
- Parameters checked in Appendix 1.6 and 2.6 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during last week of treatment (males on Day 27, females PND 4)
- Dose groups that were examined: selected animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other /see Appendices 1.1.4 - 1.1.6)

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals (at day 29)
- Maternal animals: All surviving animals (at PND 5)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special attention was paid to the organs of the reproductive system. The number of implantation sites and the number of corpora lutea were recorded in the female animals.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated in Table Section 4.7.3 were prepared for microscopic examination and weighed, respectively.

HISTOPATHOLOGY: Yes, Appendices 1.9 and 2.9 (see also Report Table 21 liiver data)
in general, five control and five high-exposed animals (male and female), occasionally 12 animals per sex (gonads and accessory organs); for the liver five control animals and 12 animals of all groups each.
The pathology report is included in Appendix 7.
Statistics:
see Report 4.8: ....The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For the high-dose P-males (5000 ppm), significantly lower body-weight values were recorded during the entire treatment period. On day 28, the mean body weight was approx. 9% lower than that of the control (Table 10 and Figure 3).

In the mid- and high-dose P-females, statistically significant bw decreases of approx. 8 and 10% were recorded at the end of mating (on day 14) and of approx. 11 and 20% at the end of the study (on PND4) (Table 10 and Figure 4).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the high-dose males and females (5000 ppm), significantly lower mean food consumption was noted.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increases (p<0.01) in the platelet count in both male and female high-dose group, but the observed mean values were within the historical control range. In conclusion, none of the above findings were clearly attributed to the test item administration.
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significant increases in the absolute and relative liver weights of the mid- and high-dose males and females. Other apparent absolute weight changes disappeared when related to body weight, therefore are not considered to be treatment-related (see Report 8.5.2, Tab. 20).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Liver enlargement in the high-dose males and females
Neuropathological findings:
no effects observed
Description (incidence and severity):
Note: relates to functional observation battery (FOB)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hepatocellular vacuolisation without pathological findings, with no signs of degeneration or necrosis or impairment of liver function. Morphological changes are considered to be an adaptive response rather than an adverse effect of OAPP exposure (see Report 8.5.3, Tab. 21).
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
97 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: analytical concentration 1065 ppm (mg/kg diet)
Key result
Dose descriptor:
LOAEL
Effect level:
337 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: analytical concentration 5235 ppm (mg/kg diet)
Key result
Critical effects observed:
no

DIET ANALYSYS and DOSES

 

Table 7: Summary of analytical results

 

Nominal concentration (ppm)

Measured concentrations from different samples (ppm)

Measured concentrations
Mean ± SD (ppm)

Measured concentration
(% of nominal)

300

1.  284.1 ± 8.3

2.  263.1 ± 13.5

3.  302.9 ± 4.0

283.4 ± 19.9

94.5%

1250

1.  1147.5 ±2 6.0

2.  1014.8 ± 7.5

3.  1031.2 ± 19.0

1064.5 ± 72.

85.2%

5000

1.  5674.6 ± 129.1

2.5194.0 ±8 2.9

3.4835.6 ± 181.0

5234.7 ± 421.0

104.7

 

Note: Set 1 was measured September 18-21 2015; Set 2 was measured October 22-24 2015; Set 3 was measured November 04-06 2015.

 

Table 8: Mean test item intake

 

OAPP concentration in diet (ppm)

Target dose levels (mg/kg bw/day)

Mean Test Item Intake (mg/kg bw/day)

 

 

Males

Females

Combined for Males and Females

300

at least 15

21.0 ± 1.2

27.5 ± 1.3

24.5

1250

at least 62.5

86.5 ± 3.4

107.7 ± 5.8

97.1

5000

at least 250

314.8 ± 13.9

360.3 ± 26.0

337.6

 

 

HISTOPATHOLOGY

 

Table 21: Microscopic examination of the livers of P0 -males and P0-females

 

 

Groups/Concentration (ppm)

 

Low-dose (300 ppm)

Mid-dose (1250 ppm)

High-dose (5000) ppm

Males (Day 29)

n=12

n=12

n=12

Hepatocellular vacuolation

3

11

12

Minimal

3

10

1

Slight

0

1

7

Moderate

0

0

4

Females (Day PP5)

n=12

n=12

n=12

Hepatocellular vacuolation

2

12

12

Minimal

1

6

0

Slight

1

4

2

Moderate

0

2

10

 

 

Conclusions:
Repeated oral dietary exposure to approximately 340 mg/kg bw/d of OAPP produced minor toxic effects (reduced body weights, body weight gain and food consumption) in male and female rats. Although considered to be related to OAPP exposure, the increased liver weights and hepatocellular vacuolation observed at the highest dietary dose was considered to be an adaptive response. Under the conditions of this study, the systemic toxicity NOAEL is considered to be 97 mg/kg bw/d.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
high
System:
other: general systemic, growth
Organ:
other: liver (not adverse under test conditions)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
duplication of group size (10 instead of 5 per sex and dose); satellite group: 5 per sex and dose.
Principles of method if other than guideline:
Group sizes were increased to improve the biostatistical power.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares LA 300 (phenol, methylstyrenated)
- Lot/batch No.: 28166
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light
Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF (Specified Pathogen Free) breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 205 +/-12 g (m); 163 +/-9 g (f)
- Fasting period before study: no
- Housing: All the study proceeded in SPF (Specified Pathogen Free) animal house of CETA under conditions according to SOP No. 12. Animals were housed in SPF animal room, 2-3 rats of the same sex (during acclimatization period) or individually (during treatment period) in one plastic cage 40x25x20cm) containing sterilised clean shavings of soft wood.
- Diet: Free access to food (ad libitum; complete pelleted diet for rats and mice in SPF breeding (ST 1 BERGMAN);
The diet was sterilised before use.
- Composition of the diet: Wheat, oats, fish meal powder, dried snail-clover, soya extracted groats, wheat sprouts, dehydrated yeast,
calcium carbonate, vitamin and mineral complex.
- Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%,
Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%.
- Water (e.g. ad libitum): Free access to drinking water; ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-3°C
- Humidity: Relative humidity of 30-70%
- Air changes (per hr): approx. 15
- Photoperiod: 12 h light, 12 h darkness

Type of coverage:
occlusive
Vehicle:
olive oil
Details on exposure:
TEST SITE
Approximately 24 hours before testing, fur was shaved from dorsal area of the trunk of the test animals.
- Area of exposure: not less than 10% of the body surface (approx. 5-6 cm x 8-9 cm)
- Type of wrap: During exposure, the test substance was held in contact with the skin with porous gauze dressing and non-irritating tape for 6 hours.
- Time intervals for shavings: approx. weekly

REMOVAL OF TEST SUBSTANCE
- The treated and control groups were treated 7 days per week at the same daytime (7.00 – 9.00 am) for the period of 28 days.
- At the end of the exposure period (6 h daily), residual test substance was removed using water.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The concentrations of emulsions at all dose levels were adjusted such as to ensure the
application of 1 ml per 100 g of body weight.
- The emulsion was prepared daily just before administration.
- Constant volume used: yes

VEHICLE
- Justification for use and choice of vehicle: Due to the viscosity, the test substance was applied as emulsion in olive oil.
- Amount(s) applied: 1 ml olive oil per 100g body weight (same as treatment groups)

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days; 6 h/day
Frequency of treatment:
daily (7 d/week)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Controls
Dose / conc.:
160 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups: 10 male, 10 female
Satellite groups: 5 male, 5 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Basis for selected doses: Range finding study with dose levels of 100, 250, 500, and 1000 mg/kg/day.
The doses for the dose-range finding experiment were derived from the limit dose of 1000 mg/kg/day (according to the guideline).
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All rats were examined for vitality or mortality daily during the treatment and recovery period.
The health condition and behavior were controlled daily during the check-in, acclimatisation period, during the administration and during the recovery period in groups.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of administration, then at weekly intervals.
During the first part of observation, behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behaviour.
The second part was the observation during removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanness of fur around foramina.

DERMAL IRRITATION (if dermal study): yes, examined

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes;
Once every week, the remainder of pellets was weighed in each cage. Average values were calculated for each week of the study.
The food consumption for each animal/day was calculated from the average values of each group.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and
body weight gain data: Yes
Weight gains were computed as arithmetic mean per group and day.

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice per week

OPHTHALMOSCOPIC EXAMINATION: not examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of test period (day 29 of study of main groups; day 42 of study of satellite groups)
- Anaesthetic used for blood collection: Yes (light ether narcosis)
- Animals fasted: Yes (18 h before blood sampling)
- How many animals: no data
- Parameters checked in table No. 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes (18 h before blood sampling)
- How many animals: no data
- Parameters checked in table No. 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of test period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: no data
- Parameters checked in table No. 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Daily at the end of the exposure period (main groups) or at the end of the recovery period (satellite groups)
- Dose groups that were examined: all groups
- Battery of functions tested: The sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated, and motor activity assessment was conducted. Moreover, the individual observations of grip strength were performed using dynamometer. Measurements were made on: 1) forelegs, 2) hindlegs, 3) all four legs.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy of the cranial, thoracic and abdominal cavities; organs were collected for weighing and further histological examination.
(absolute and relative weights of lungs, liver, kidneys, adrenals, testes, epididymides, brain and pituitary gland)

HISTOPATHOLOGY: Yes; organs of control animals and animals treated with the highest dose level (1000 mg/kg/day) were examined:
Normal and treated skin, lung, liver, kidneys, spleen, brain, pituitary gland, testes, epididymides (both fixed in Davidson solution), as well as ovaries
and accessory genital organs, adrenal glands, heart and target organs (organs showing gross lesions or changes in size).
Statistics:
ANOVA test - Analysis of Variance (QC.Expert 2.5) - at the significance level of 0.05 was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. The respective control groups with vehicle were compared with the three treated groups, and the respective satellite controls with vehicle were compared with the treated satellite groups.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
incidental effect, no mortality
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
see details below (clinical signs)
Mortality:
mortality observed, treatment-related
Description (incidence):
incidental effect, no mortality
Body weight and weight changes:
no effects observed
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):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
see details below: variations in parameters are minor and sporadic, unrelated to dose.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
see below for details
Urinalysis findings:
no effects observed
Description (incidence and severity):
see below for details
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
see below details
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below for details: minor, not toxicologically relevant
Gross pathological findings:
no effects observed
Description (incidence and severity):
see below for details: Sporadic changes noted, none in recovery groups
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
see below for details: Only in females changes in kidney and urethra, not considered treatment-related.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
no key endpoint
Details on results:
CLINICAL SIGNS AND MORTALITY:
Males: One animal at the dose level of 400 mg/kg/day showed red secretion around the nose or eyes.
In one animal at the dose level of 160 mg/kg/day diarrhoea was observed.
Slight skin erythemas of the application area were recorded at the dose level of 400 mg/kg/day (one male in the 3rd week), and marked erythemas at the dose level of 1000 mg/kg/day (1 male in the 1st week, 4 males and 2 satellite males in the 3rd week and 2 males and 1 satellite male in the 4th
week).
Females: At the dose level 400 mg/kg/day, slight skin erythemas of the application area were recorded only in one animal in the 3rd week.
At the dose level of 1000 mg/kg/day, this change of application area was observed in a few females during the whole application:
The following number of affected females during the test period showed dermal irritation: 6 females and 1 satellite female in the 1st week, 1 female in the 2nd week, 5 females and 3 satellite females in the 3rd week, and 3 females and 2 satellite females in the last week of application.
No animal died during the test period.

BODY WEIGHT AND WEIGHT GAIN:
In both sexes no significant differences between the treatment groups and the control groups were observed during the whole test period.

FOOD CONSUMPTION:
In both sexes no significant differences between the treatment groups and the control groups were observed during the whole test period.

FOOD EFFICIENCY:
Males: During the 1st week of application, a slightly decreased food efficiency was recorded at the lowest dose level. From the 2nd week to the 4th
week of application, the food efficiency revealed no significant differences at all dose levels compared with the control group.
Females: In the 1st week of application, an increased food efficiency was recorded at all dose levels. Relatively well-balanced conversion was recordedfrom the 2nd and 3rd week of application. In the last week of application, a decrease in food efficiency was recorded at all dose levels.
Satellite groups: In the 1st week of the test period, the food efficiency was increased in the treated group of both sexes. From the 2nd week to the end of the application period and during the recovery period no sicnificant differences were observed.

WATER CONSUMPTION:
The average water consumption of the treated groups of both sexes revealed no significant differences in comparison with the control groups during the whole application period except in the female group of the lowest dose level, it was lower than in the control group during the whole study.
Satellite males: The water consumption of both groups was not signifcant different during the whole application and recovery period.
A slight decrease was recorded only in the 3rd week of application.
Satellite females: In the treated group, the water consumption was higher than in the control group during the whole application period and the two weeks of recovery (the increase especially during treatment can be explained by the higher mean body weight of the treated group).

HAEMATOLOGY: see Report, Table 14 and 15
Males: Changes in the haemocoagulation system were recorded. A slightly shortened prothrombin time (PT) was measured at the dose levels of 400 and 1000 mg/kg/day with statistical significance only at the middle dose.
The concentration of fibrinogen was statistically significantly increased at the dose level of 1000 mg/kg/day.
Total leucocyte count was slightly decreased (without statistical significance) at all dose levels.
Other measured parameters were similar to the control group.
Females: A shortened prothrombin time (PT) was measured at all dose levels, but with statistical significance only at the dose level of 400 mg/kg/day.
Total leucocytes count was decreased with statistical significance at the dose level of 160 mg/kg/day.
A slightly decreased mean corpuscular volume was recorded at all treated groups but without statistical significance.
Other measured parameters were similar to the control group.
Satellite groups: A statistically significant increase in the value of haematocrit was recorded in the treated group in both sexes.
Other parameters of red blood cell - total erythrocyte count and MCV - were slightly increased without statistical significance.
A statistically significant decrease in the platelet count was recorded in the treated females.
Total leucocyte count of treated males was insignificantly decreased.
Fibrinogen concentration of the treated males was statistically significantly increased.
A shortened PT was recorded without statistical significance. All other measured parameters were similar to the control group.

CLINICAL CHEMISTRY: see Report, Table 16 and 17
In both sexes an increased activity of ALP was recorded at the dose levels of 400 (males) and 1000 mg/kg/day (males and females), but with
statistical significance only at the high dose level.
All treated groups showed an increased value of bilirubin without statistical significance and without evidence of a dose-response relationship
(in females only at the dose level of 1000 mg/kg/day).
In males at the dose level of 160 mg/kg/day, a significantly decreased concentration of total protein together with an insignificantly decreased content of albumine was measured. In females the total protein content was significantly decreased at the dose levels of 160 and 400 mg/kg/day.
Only in male animals a significantly increased concentration of glucose was observed.
Only in females a decrease of ALT activity (significant at the dose levels of 160 and 1000 mg/kg/day), a decrease of anorganic phosphorus
(signifcant at the dose level of 1000 mg/kg/day), and a slight increase of the value of urea at the dose levels of 400 and 1000 mg/kg/day (without
significance) were determined.
All other measured parameters were similar to those of the control groups.

Satellite groups:
Males: A significantly decreased value of urea was recorded in the treated group. The apparent effect is not treatment-related but due to
the inexplicably high mean urea level observed in the urine of the concurrent control group.
Furthermore, a significantly increased activity of AST, a decreased activity of ALT an increased concentration of bilirubin, an increased concentration of chloride and a decreased concentration of potassium with statistical significance was recorded in the treated group.
Females: A statistically significant decrease in the concentration of calcium and potassium were recorded in the treated group.
The value of total protein in the treated group was slightly decreased (without statistical significance).
All other measured parameters were similar to the control group. All parameters were within the historical ranges of average control values.

URINALYSIS: see Report, Table 18 and 19
No statistically significant changes in urine were recorded at any dose level, except a statistically significant decrease in urine pH of females at the
dose level of 160 mg/kg/day.
Presence of protein, blood and leucocytes was recorded in the urine of one male at the dose level of 400 mg/kg/day.
Presence of protein in the urine of two males and leucocytes in the urine of one male were detected at the dose level of 1000 mg/kg/day.
The urine of three males at the highest dose level showed white clouding.
Satellite groups: No significant intergroup differences were observed. The urine of one treated male showed blood.
Presence of leucocytes was recorded in the urine of three control and two treated males.

NEUROBEHAVIOUR: see Report, Table 12 and 13 (Functional observation)
The reaction to approach, touch, the reaction to noise, and the reaction to pain and light (pupillary reflex) were the same in all treated groups of males and not different from the control groups. The values of grip strength of forelegs and hind legs of all groups showed no significant differences.
All inter- and intra-group differences in scores were considered to be a result of normal variation for the rats of the used strain and of age, and were of no toxicological importance.

ABSOLUTE ORGAN WEIGHTS:
In male animals at the dose levels of 400 and 1000 mg/kg/day, the absolute weight of the pituitary gland was significantly increased. Male animals at
these dose levels also showed a slightly increased weight of liver (not significant).
Female animals showed a slightly increased weight of liver at the dose level of 400 mg/kg/day.
Further significant intergroup differences were not observed.
Satellite groups: The treated males showed a decreased body weight at the end of the recovery period. Hence, the absolute weight of all organs was
slightly lower than in the control males, but only the absolute weight of epididymides in treated males was significantly decreased.
In females no significant differences were observed.

RELATIVE ORGAN WEIGHTS:
In male animals a statistically significant dose-dependant increase in the relative weight of liver was recorded at the dose levels of 400 and
1000 mg/kg/day.
Also the relative weight of the pituitary gland was significantly increased at the dose levels of 400 and 1000 mg/kg/day.
Female animals at the dose levels of 400 and 1000 mg/kg/day showed as well a slightly increased relative weight of liver (not significant).
Further significant intergroup differences were not observed.
In the satellite groups no significant deviations were found.

GROSS PATHOLOGY: see Report, Table No. 24 and 25 for details
The macroscopic examination revealed few pathologic changes in both sexes in all groups.
In the satellite groups no macroscopic recognisable pathologic changes were found.

HISTOPATHOLOGY: NON-NEOPLASTIC: see Report, Table 26 and 27 for details
At the application area no significant changes were found in all examined groups and both sexes.
Only in female animals, the most frequent changes were corticomedullary mineralisation of the kidneys and hydrometra of the uterus, without evidence of an association to treatment, since there was a remarkable background of these effects, while reduced or absent in the recovery groups (see also Attached Document, Table 26 and 27).


Dose descriptor:
NOEL
Remarks:
(highest dose tested)
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall systemic effects
Critical effects observed:
not specified
Conclusions:
The dermal administration of the test substance Novares LA 300 to rats for a period of 28 consecutive days did not cause unacceptable pathological changes at the site of administration. No significant changes, which indicate organ dysfunction, were observed in clinical biochemistry, haematology and urinalysis parameters, and in histopathological examination at any dose level.
Based on these results, 1000 mg/kg/day, the highest dose tested, can be considered as a dermal NOEL(28d, systemic) exhibiting no toxicologically significant effects in male and female rats.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Under the conditions of the subchronic test conditions (OECD 408 combined with OECD 443 (continuous dietary dosage for 100 days to young rats after weaning), OAPP failed to produce any specific toxicity, although the liver proved to be the primary target organ: This was expressed in dose-related increases in liver weight and hepatocellular vacuolation in both sexes. There was no evidence of relevant primary or secondary effects on clinical-biochemical, haematological parameters as well as TSH- and T4-hormone status. Given the observation that the liver changes were absent in the 30d-recovery groups, they are considered to represent a non-adverse, adaptive metabolic response on the chemical body burden.

It was conspicuous that the body-weight development was significantly retarded in high-dose males and females, more pronounced in the latter, by 9 % and 18 %, respectively. The effect in females is considered to be adverse, based on the relatively high decline as compared to the control. However, food consumption was also distinctly reduced in this exposure group, which was not evident in the other treated groups. Hence, this may have inhibited growth to a certain extent.

Human relevance: The mode of action on the liver can be anticipated to be principally valid for humans. However, not any treatment-related impact was evident after repeated dermal exposure, which is supposed to be the more important potential route of human exposure. Hence, based on the outcome from the dermal 28d-study, the relevance of the hepatic effects for humans, including workers, is very low.

Additional information

Regarding the repeated-dose toxicity of ‘Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol' (OAPP) (previous name: phenol, methylstyrenated), one sub-chronic study (100d) and two subacute studies (28d) are available.

1.) Oral medium-term repeated dose toxicity study

Repeated-dose toxicity was tested under GLP conditions in a study according to OECD TG 443 (Extended One-Generation-Reproduction Toxicity Study, EOGRTS), combined with OECD 408). Doses were 150, 500 and 1500 mg/kg diet (nominal) corresponding to 12, 40, and 122 mg/kg bw/day based on food consumption and measured concentrations of the test substance in the diet. At the highest dose, effects on the F1 -females were observed (reduced body weight gain, and food consumption), which was considered to be adverse (see also Mode of Action). Although related to OAPP exposure, the increased liver weights and hepatocellular vacuolation observed at the highest dietary dose was considered to be an adaptive response. Under the conditions of this study (Hargitai 2017), the systemic toxicity NOAEL(100d) is considered to be 40 mg/kg bw/day, based on inhibited body-weight development.

2.) Oral short-term repeated dose toxicity study:

Repeated-dose toxicity was tested under GLP conditions in a study according to OECD TG 422 (Combined Repeated Dose Toxicity study with Reproduction / Developmental Toxicity Screening Test). Doses were 300, 1250, and 5000 mg/kg diet (nominal) corresponding to 24, 97, and 337 mg/kg bw/day based on food consumption and measured concentrations of the test substance in the diet. At the highest dose, effects on the parental (P0) animals were observed (reduced body weights, body weight gain, and food consumption). Although considered to be related to OAPP exposure, the increased liver weights and hepatocellular vacuolation observed at the highest dietary dose was considered to be an adaptive response. Under the conditions of this study (Hargitai 2016), the systemic toxicity NOAEL(28d) is considered to be 97 mg/kg bw/day,

3.) Dermal short-term (28 d) repeated dose toxicity study:

The study was conducted under GLP conditions with OAPP according to OECD TG 410, but using an increased number of test animals per dose (male and female rats, 10 each instead of 5). The test substance was applied in olive oil at doses of 160, 400, and 1000 mg/kg bw/day. Two satellite groups for recovery were included (control and high dose 1000 mg/kg bw/day, 5 animals per sex each). Slight dose-related increases in the relative liver weights may provide some evidence of a substance-dependent systemic effect. No other toxicologically significant changes, which indicate organism dysfunction, were observed in clinical biochemistry, haematology, urinalysis parameters, histopathological and behavioural examinations at any dose levels. Apparent effects noted were sporadic and considered as casual. The 28d NOAEL (systemic effects) for male and female rats was the highest dose tested and corresponds to 1000 mg/kg bw/day (Plodikova 2012).

Justification for classification or non-classification

No classification needed: The effects observed are unspecific: the increase in liver weights and slight to moderate hepatocellular vacuolation without histologic manifestation after recovery are considered to be an adaptive response. On the other hand, the test item-related growth retardation/inhibition without obvious toxic symptoms was assumed to be adverse, yet not sufficient for classification.