Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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

Cas Number:

68512-30-1

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:

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

Doses / concentrations

Doses / concentrations 1

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)

Doses / concentrations 2

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)

Doses / concentrations 3

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)

Doses / concentrations 4

Dose / conc.:

283 other: ppm (analytical)

Remarks:

Low concentration in diet - analytical (see Report Tab. 7)

Doses / concentrations 5

Dose / conc.:

1 065 other: ppm (analytical)

Remarks:

Mid concentration in diet - analytical (see Report Tab. 7)

Doses / concentrations 6

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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

1

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)

2

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)

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

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

 

 

Applicant's summary and conclusion

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.