Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

GLP compliance:

yes (incl. QA statement)

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
- Expiry date: 2017-02-13
- 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 or test system 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: at sacrifice (males on day 29, females on PND 5)
- 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 liver 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/day 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/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol

EC Number:

700-960-7

Molecular formula:

not applicable

IUPAC Name:

Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol

Test material form:

liquid: viscous

Details on test material:

- Manufacturers identification: Novares LA 300
- Substance type: organic
- Test material is 'Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol' (OAPP), EC list number 700-960-7 (assigned manually to validated substances from inquiries by ECHA). Originally the substance phenol, methylstyrenated, CAS No. 68512-30-1, EC No. 270-966-8 was submitted for registration. Subsequent to substance validation, the identity of the substance was changed by ECHA.
- for additional information see respective study record

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

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

corn oil

Remarks:

as carrier for mixing with the diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): once
- Mixing appropriate amounts with 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 in corn oil.
- Concentration in vehicle: depending on dose group
- Amount of vehicle: 4% in the diet

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation, up to 14 days
- Proof of pregnancy: sperm in vaginal smear or vaginal plug referred to as day 0 of pregnancy (GD0)
- Any other deviations from standard protocol:

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: pre-mating 14 days and mating/post-mating at least 14 days
Females: pre-mating 14 days, mating up to 14 days, gestation 22-24 days, and lactation 4 days

Frequency of treatment:

continuous

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 ppm (nominal)

Remarks:

actual dose received: 24 mg/kg bw/day (calculated) (see Report Tab. 8);
Low dose - estimated target ~15 mg/kg bw/d (via diet)

Doses / concentrations 2

Dose / conc.:

1 250 ppm (nominal)

Remarks:

actual dose received: 97 mg/kg bw/day (calculated) (see Report Tab. 8);
Mid dose - estimated target ~62.5 mg/kg bw/d (via diet)

Doses / concentrations 3

Dose / conc.:

5 000 ppm (nominal)

Remarks:

actual received dose: 337 mg/kg bw/day (calculated) (see Report Tab. 8);
High dose - estimated target ~250 mg/kg bw/d (via diet)

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) 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

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 1x/d
- Cage side observations checked in Appendix 1.1.1 were included.

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

BODY WEIGHT: Yes
- 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
- 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

OTHER: Functional observation battery (FOB) including grip strength and motor activity - with selected animals during the last week of treatment: (males on Day 27, females PND 4)

OTHER: Haematology and urinalysis (at sacrifice - males on day 29, females on PND 5)

Oestrous cyclicity (parental animals):

During the mating period until a sperm positive vaginal smear or a vaginal plug was identified.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]

Special attention was paid to evaluation the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. (4.7.3)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other: body weight

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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals after mating
- Maternal animals: all surviving animals after weaning, terminally one day after the last treatment (4.7)

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 (4.7.3)
The tissues indicated in the Table were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

not applicable

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.

Reproductive indices:

see Report Tab.14 and 15: Summary of reproductive parameters / Table 16 Summary of the intrauterine evaluation)

Offspring viability indices:

Report 4.8:
- Mean pup body weight (per pup within the group and per litter) on PND 0 and 4 †
- Mean pup body weight gain (per litter) between postnatal days 0-4 †
- Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4 †
- * Survival Index of pups on postnatal days 0 and 4 †
- *Sex distribution % (on postnatal days 0 and 4) †

[see also Report Tab.16 (Summary of the intrauterine evaluation) and Table 17 (Summary of clinical observation of the pups)]

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 (Report, 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) (Report, 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, the 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

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:

not specified

Description (incidence and severity):

not applicable

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

see Appendix 1.1.7

Reproductive function: sperm measures:

not specified

Description (incidence and severity):

see Report 4.7.3

Reproductive performance:

no effects observed

Description (incidence and severity):

Test item-related adverse effects were seen in the reproductive parameters of the high-dosed P0-females: significantly decreased number in corpera lutea and in implantations (Report summary). Note: Final clarification on toxicological significance needed (see Report Tab.16).

Else, no effects: Female mating index, female fertility index, and female gestation index were 100% across all groups, but the fertility index in the control group was 83% (see Report, Table 15). All male mating and fertility indices were 100% (see Table 14).

Details on results (P0)

No toxicologically significant differences were detected in pre/post-implantation loss, intrauterine, post-natal or total mortality values in any of the test item-treated groups (Report summary).

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: highest dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Cannibalism caused the loss of one toal litter of the high-dose dams, which supresses the post-natal survival index by about 6 - 7 % compared with the control (Report, Table 17), while the numbers of viable pups on PND4 and the pup survival indices on PND0 and PND4 in the Low and Mid dietary exposure groups (300 and 1250 ppm) were comparable to control values. Since low maternal body weight and food intake affects lactation, the reduction in viable pups at PND 4 at the High exposure level was not unexpected and was considered to be secondary to maternal toxicity.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the high-dose group, there is some not statistically significant evidence of growth retardation from PND0 to PDN4 (average -6.5%), the corresponding mean body-weight gain was about 21 % less than that in the control group (Report Tab. 18). No effects evident in F1 of the low- and mid-dose group.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

not applicable. Suckling was normal but one control pup failed to.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Only external macroscopic examinations performed: One pup each with haemorrhage was recorded in the Control, Mid and High dose groups (Report 8.4.1.)

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

Remarks on result:

other: post-natal development until PND4 includes notable cannibalism

2

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

Remarks on result:

other: post-natal development until PND4 includes notable cannibalism

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

other: unspecific post-natal impairment of viability

Organ:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

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

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

DIET ANALYSIS and DOSES

Table7: 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±26.0 2.    1014.8±7.5 3.    1031.2±19.0	1064.5±72.3	85.2%
5000	1.    5674.6±129.1 2.    5194.0±82.9 3.    4835.6±181.0	5234.7±421.0	104.7%

Note: Set1 was measured 18-21 September 2015; Set2 was measured 22-24 October 2015; Set3 was measured 04-06 November 2015.

Table8: Mean test item intake

OAPP concentration in diet (ppm)	Target dose levels (mg/kgbw/day)	Mean Test Item Intake (mg/kgbw/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

Table21: Microscopic examination of the livers of P0 -males and P0-females

	Groups/Concentration (ppm)
	Low-dose (300)	Mid-dose (1250)	High-dose (5000)
Males (Day29)	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 PN5)	n=12	n=12	n=12
Hepatocellular vacuolation	2	12	12
Minimal	1	6	0
Slight	1	4	2
Moderate	0	2	10

Applicant's summary and conclusion

Conclusions:

The number of pups born and live-born as well as post-natal weight gain were significantly lower in the High dietary exposure group (5000 ppm; actual dose received: 337 mg/kg bw/day). These effects were considered secondary to maternal toxicity (body weight differences), with no indication of any specific reproductive toxicity at any exposure dose level. Note: this evaluation is provisional: The viability aspect depends on the interpretation of the role of cannibalism, which caused the loss of a total litter, while post-natal groth retardation is independent of this factor.