1-phenylethyl acetate

Carcinogenicity

Description of key information

Using data from benzyl acetate as read-across, 1 -phenylethyl acetate is not predicted to be carcinogenic

KEY STUDY REPORT
NTP (1993) NTP TR 431 Toxicology and Carcinogenesis Studies of Benzyl Acetate (CAS NO. 140-11-4) In F344/N Rats and B6C3F1 Mice (Feed Studies). This report contains a two year study on both mice and rats conducted to a method similar to OECD 451 and in line with GLP.
SUPPORTING STUDY REPORT
NTP (1986) 'NTP Technical Report on the Toxicology and Carcinogenesis Studies of Benzyl Acetate (CAS No. 140-11-4) in F344/N rats and B6C3F1 mice (gavage studies)'. This report contains a two year study on both mice and rats conducted to a method similar to OECD 451 but not in line with GLP.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

Study commenced August 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid study is available for the analogue substance benzyl acetate. It is conducted in compliance with good scientific principles, with no or minor deviations from standard protocols. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Principles of method if other than guideline:

The general principles of the methodology are similar to method OECD 451, minor deviations are apparent, such as dosing five days per week instead of seven, which the guideline states may be acceptable and only two dosing levels rather than the recommended three were employed.

GLP compliance:

not specified

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Breeding Laboratories, Portage, MI- Age at study initiation: 8 weeks- Housing: Polycarbonate, Lab Products Inc, Garfield, NJ, USA- Diet (e.g. ad libitum): Wayne Lab-Blox, Allied Mills Inc, Chicago ad libitum- Water (e.g. ad libitum): tap water ad libitum- Acclimation period: 2 weeksENVIRONMENTAL CONDITIONS- Temperature (°C): 21-24 ºC- Humidity (%): 30-60%- Air changes (per hr): 15 per hour- Photoperiod (hrs dark / hrs light): 12 hours of fluorescent light per day

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:- Doses were prepared on a weight-to-volume basis by pipetting the appropriate amount of benzyl acetate into a vessel and adding enough corn oil to give the desired concentration. Solutions were mixed until they were visually homogenous. Once prepared, mixtures were stored at 5 °C for no more than 11 days.VEHICLE- Justification for use and choice of vehicle (if other than water): Although exposure to benzyl acetate is primarily in food, the study opted to dose in corn oil via gavage due to the substance’s volatility and it’s reactivity with moisture present in feed.- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of benzyl acetate in corn oil were selected at random and analysed periodically at Southern Research Institute.Method used until 04.04.79:Samples of benzyl acetate were received as corn oil mixtures. 0.5 ml aliquots were dissolved in 10 ml chloroform. Analysis was by vapour-phase chromatography under the following conditions:Instrument: Perkin-Elmer 910Detection: Flame ionizationColumn: 3% OV-17 on 80/100 Suplecoport, 1.8 m x 4 mm I.D., glassTemperatures: Inlet, 140 ºC; Oven, 100 ºC, isothermal; Detector, 170 ºCInjection Size: 2 µlRetention time: 2.8 minThere was no correction for workup loss since samples were injected without any extraction or workup procedure. The gavage samples were compared with the reference standards of benzyl acetate prepared volume/volume in corn oil, dissolved in chloroform in the same manner as the gavage samples and analysed under the same chromatographic conditions.Method used after 04.04.79:Samples of benzyl acetate were received as corn oil mixtures in sealed syringe bottles. Samples were extracted with methanol for 3 minutes (20 ml methanol with 0.5ml of sample made up in corn oil). Analysis was by vapour-phase chromatography under the following conditions:Instrument: Sigma 1Detection: Flame ionizationColumn: 3% OV-17 on 80/100 Suplecoport, 1.8 m x 4 mm I.D., glassTemperatures: Inlet, 140 ºC; Oven, 100 ºC, isothermal; Detector, 170 ºCCarrier gas: HeliumInjection Size: 1 µlRetention time: 2.5 minThe gavage samples were compared with the reference standards of benzyl acetate prepared volume/volume in corn oil then extracted with methanol in the same manner as the sample. There was no correction applied to the samples since samples and reference standard were treated in the same manner.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

5 days per week

Post exposure period:

Not applicable, terminal kill was performed week 104-106.

Remarks:

Doses / Concentrations:0, 500 and 1000 mg/kg bwBasis:

No. of animals per sex per dose:

Fifty

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: on the basis of the results from the 13 week study

Observations and examinations performed and frequency:

MORTALITY AND MORBIDITY: Yes- Time schedule: twice daily. Animals that were judged to be moribund were killed with carbon dioxide and necropsied.DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: at each weighing period - weekly for first 13 weeks, monthly until week 91 and then every two weeksBODY WEIGHT: Yes- Time schedule for examinations: every week for the first 13 weeks and monthly thereafterFOOD 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: NoWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): NoOPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: NoCLINICAL CHEMISTRY: NoURINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: YesNecropsies were performed on all animals found dead and on those killed at the end of the study unless precluded in whole or in part by autolysis or cannibalisation.HISTOPATHOLOGY: YesThe following organs wee examined microscopically gross lesions, brain, pituitary, thymus, spleen, thyroid, parathyroid, lung and bronchi, trachea, heart, oesphagus, stomach (pylorus and fundus), duodenum, jejunum, ileum, large intestine, pancreas, adrenal, kidney, liver, gallbladder, skin, mammary gland, urinary bladder, prostate, seminal vesicles, testes or uterus/ovaries, femur with marrow, abnormal lymph nodes, salivary gland, thigh muscle, sciatic nerve, costochondral junction, larynx, mesenteric lymph nodes, nasal cavity and spinal cord

Other examinations:

Animal disease condition at the laboratory was monitored by the use of sentinel animals. Plasma samples were obtained from these animals (5 per sex and species) at 6, 12, and 18 months and from vehicle control animals at 24 months. These samples were assayed for the various viral serology titers.

Statistics:

SURVIVAL ANALYSISThe probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented graphically. Animals were censored from survival analysis at the time they were found dead of other than natural causes. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups of equality and Tarone's (1975) extensions of Cox's method for testing a dose-related trend. All reported P values are two-sided.CALCULATION OF INCIDENCE RATESThe incidence of neoplastic or non-neoplastic lesions has been given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which the site was examined. In most instances, the denominators include those animals for which the site was examined histologically. However, when macroscopic examination was required to detect lesions prior to histologic sampling, or when lesions could have appeared at multiple sites, the denominators consist of the number of animals on which necropsies were performed.HISTORICAL CONTROL DATAAlthough the concurrent control group is always the first and most appropriate control group used for decision-making, there are certain instances in which historical control data can be helpful in the overall evaluation of tumour incidence. Consequently, control tumour incidence from the NTP historical control database (Haseman et al., 1984) are included for those tumours in these studies appearing to show substance-related effects.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

MORTALITYThe survival of the high-dose group of females was significantly increased when compared to that of the control groups. No other significant differences in survival were observed between any groups of either sex. Two low-dose males, one high-dose male, four low-dose females and one high-dose female were accidentally killed and these animals were censored from the statistical analysis of survival at the date of death. In males, 38/50 (76%) of the controls, 33/50 (66%) of the low-dose group and 39/50 (78%) of the high-dose group lived to termination. In females, 15/50 (30%) of the controls, 18/50 (36%) of the low-dose group and 30/50 (60%) of the high-dose group lived to termination. The survival data include one low-dose male that died during termination and this animal was considered to have been killed at the end of the study.BODY WEIGHT AND WEIGHT GAINThroughout the study, mean bodyweights of dosed and control animals were comparable. After week 20, mean bodyweights of low- and high-dose females were slightly greater than those of the controls.HISTOPATHOLOGYHepatocellular adenomas occurred in mice of each sex with statistically significant positive trends, and the incidences in the high-dose groups were greater than those in the controls. Hepatocellular carcinomas were marginally elevated in dosed males and high-dose females.Squamous cell papillomas or carcinomas of the forestomach occurred with a positive trend in males. The incidence of these tumours was also marginally increased in the high-dose females. The incidences of these tumours both in high-dose males and high-dose females were considerably higher than the historical corn oil gavage control rates at the testing laboratory and throughout the program. Forestomach hyperplasia occurred at increased incidence in dosed animals of either sex.ANALOGUE APPROACH JUSTIFICATION:- See attached “Justification for read-across” document for full details.- In summary, important considerations for the use of read-across for acute toxicity are: i) 1-phenylethyl acetate (the target chemical) has similar physico-chemical properties as benzyl acetate (the source substance), ii) there are structural similarities between the two chemicals, iii) the OECD QSAR Toolbox assigns an identical toxicity profiles to both chemicals, and iv) both chemicals have been tested for acute oral toxicity, which demonstrated that neither substance requires classification for acute toxicity and that the benzyl acetate will represent a worst-case scenario, and are adequate for classification and labelling and risk assessment purposes. The information reported in this summary is included to demonstrate comparability between the source (benzyl acetate) and target (1-phenyl-ethyl acetate) substance.

Relevance of carcinogenic effects / potential:

Under the conditions of the study, some evidence of carcinogenicity was observed in both male and female mice . An increased incidence of hepatocellular adenomas and squamous cell neoplasms of the forestomach were observed. However reassessment of the conclusions of the study in the 1993 study concluded that the lesions noted were confounded by the dosing method. The positive results observed in the gavage study were considered to have resulted from the method of dosing rather than the carcinogenic potential of the test substance. The squamous cell neoplasms of the forestomach were not observed in the feed study and the difference is attributed to the deposition of the test substance during the gavage administration into the stomach. Furthermore, the occurrence of hepatocellular adenomas were not observed in the feeding study. The different dosing methods are considered to greatly increase the circulating levels of the test substance and its metabolites, and potentially saturate the benzoic acid elimination pathway. Overall, from the combined reports, it was concluded that benzyl acetate did not demonstrate any evidence of carcinogenic activity.

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects were noted at all dosing levels

Remarks on result:

not determinable

Remarks:

no NOAEL identified. Effect type:carcinogenicity

Table 1: Mean bodyweights and survival of mice

	Control		500 mg/kg			1000 mg/kg
Weeks on study	Average wt (g)	No. of survivors	Average wt (g)	Wt (% of controls)	No. of survivors	Average wt (g)	Wt (%) of controls	No. of survivors
Male
0	24.1	50	24.2	100	50	24.2	100	50
1	25.1	50	25.8	103	50	26.3	105	50
2	27.2	50	27.6	101	50	27.7	102	50
3	27.9	50	28.6	103	50	28.3	101	50
4	28.4	50	29.3	103	50	28.9	102	50
5	29.8	50	30.1	101	50	29.7	100	50
6	30.5	49	30.7	101	50	30.7	101	50
7	31.4	49	31.7	101	50	31.7	101	50
8	31.5	49	31.7	101	50	32.1	102	50
9	31.5	49	32.2	102	50	32.4	103	50
10	32.9	49	32.2	98	49	32.6	99	50
11	33.8	49	33.4	99	49	33.6	99	50
13	33.5	49	33.7	101	49	33.4	100	50
14	34.4	49	34.1	99	49	33.6	98	50
19	34.3	49	36.3	106	48	36.2	106	50
22	36.5	49	37.3	102	48	36.9	101	50
26	38.6	49	39.4	102	48	39.5	102	48
31	41.2	46	41.3	100	48	41.8	101	48
35	42.5	45	43.8	103	48	42.9	101	48
40	43.8	44	44.6	102	47	44.1	101	48
44	44.0	44	44.7	102	47	44.3	101	48
49	43.8	44	44.3	101	46	43.8	100	48
53	46.2	44	47.1	102	45	46.0	100	48
57	46.1	44	46.5	101	45	46.7	101	48
61	45.3	44	45.7	101	45	45.7	101	48
66	45.1	44	46.3	103	45	45.4	101	47
71	45.1	43	45.0	100	44	46.4	103	47
75	45.7	42	45.6	100	43	46.3	101	46
79	44.5	42	45.2	102	43	46.4	104	46
83	45.4	42	45.3	100	43	46.6	103	46
87	45.3	41	45.1	100	43	45.9	101	46
93	44.7	39	43.5	97	37	45.4	102	42
97	46.3	38	45.0	97	35	46.2	100	41
102	44.8	38	41.7	93	33	44.1	98	40
104	44.5	38	42.0	94	33	43.3	97	39
Female
0	19.6	50	19.7	101	50	19.4	99	50
1	18.7	50	19.9	106	50	20.8	111	50
2	21.7	50	21.4	99	50	21.4	99	50
3	22.2	50	22.2	100	50	22.1	100	50
4	22.4	50	22.1	99	50	22.7	101	50
5	22.7	50	22.8	100	48	23.2	102	50
6	24.1	50	23.4	97	48	23.8	99	50
7	24.0	50	23.9	100	48	24.3	101	50
8	23.7	50	24.2	102	48	24.4	103	50
9	24.3	50	24.0	99	47	24.7	102	50
10	24.6	50	24.7	100	47	25.1	102	50
11	25.0	50	25.3	101	47	25.5	102	50
13	25.3	50	25.1	99	47	25.4	100	50
14	25.5	50	25.2	99	47	25.5	100	50
19	25.2	49	26.0	103	47	27.6	110	50
22	26.7	48	27.6	103	47	28.1	105	48
26	28.8	48	29.1	101	47	30.1	105	48
31	30.1	48	30.9	103	47	31.4	104	48
35	32.3	48	32.2	100	47	33.3	103	48
40	32.2	47	33.9	105	46	34.1	106	48
44	33.6	47	34.9	104	45	34.9	104	48
49	33.8	47	34.8	103	45	35.5	105	48
53	35.1	47	37.7	107	44	37.8	108	48
57	35.6	46	38.0	107	42	38.0	107	47
61	35.5	46	37.6	106	42	38.0	107	47
66	34.3	44	36.9	108	42	38.3	112	46
71	34.7	40	38.6	111	40	38.5	111	44
75	34.6	38	40.1	116	35	39.9	115	42
79	33.8	34	40.2	119	33	40.1	119	40
83	35.7	31	40.2	113	30	41.1	115	38
87	34.4	28	39.8	116	29	40.9	119	36
93	34.9	20	36.7	105	27	40.8	117	33
97	35.1	17	37.4	107	25	42.3	121	30
102	34.6	15	36.5	105	20	39.9	115	30
104	34.8	15	36.9	106	18	38.8	111	30

Table 2: Survival of mice

	Vehicle control	500 mg/kg	1000 mg/kg
Male
Animals initially in study	50	50	50
Natural death or moribund sacrifice	12	14	10
Accidental death or missing	0	3	1
Scheduled or terminal sacrifice	38	33	39
Survival P values ^(a)	0.628	0.855	0.714
Female
Animals initially in study	50	50	50
Natural death or moribund sacrifice	35	28	19
Accidental death or missing	0	4	1
Scheduled or terminal sacrifice	15	18	30
Survival P values ^(a)	0.033	0.313	0.005

^(a) The vehicle control column contains the result of the life table trend test; the columns for dosed groups contain the life table pairwise comparisons with the vehicle controls.

Pathology and Statistical Analyses of Results

Liver

In males, hepatocellular adenomas occurred with a statistically significant positive trend. Results of pairwise comparisons with the controls were significant for both the low- and high-dose groups. Adenomas or carcinomas (combined) in males also occurred with a significant positive trend and the results of pairwise comparisons were significant for the high-dose group. In females, adenomas were observed in a statistically significant positive trend. The results of pairwise comparisons with the controls were significant for the high-dose group. Adenomas or carcinomas (combined) occurred with a significant positive trend and the results of pairwise comparisons with the controls were significant for the high-dose group. One additional neoplasm was observed in the liver of a high-dose male. However, because this neoplasm was necrotic as a result of obstructed blood vessels, it was not possible to determine whether this neoplasm was an adenoma or carcinoma.

Table 3: Analysis of liver tumours in mice

	Vehicle control	500 mg/kg	1000 mg/kg
Males
Adenoma
Overall	0/50 (0%)	5/49 (10%)	13/50 (26%)
Adjusted	0%	13%	33.3%
Terminal	0/38 (0%)	3/33 (9%)	13/39 (33%)
Life Table Test	P<0.001	P=0.030	P=<0.001
Incidental Tumour Test	P<0.001	P=0.023	P<0.001
Carcinoma
Overall	10/50 (20%)	14/49 (29%)	12/50 (24%)
Adjusted	24.3%	35.9%	25.8%
Terminal	7/38 (18%)	9/33 (27%)	5/39 (13%)
Life Table Test	P=0.427	P=0.183	P=0.463
Incidental Tumour Test	P=0.536	P=0.379	P=0.548N
Adenoma or Carcinoma^(b)
Overall	10/50 (20%)	18/49 (37%)	23/50 (46%)
Adjusted	24.3%	45.1%	49.8%
Terminal	7/38 (18%)	12/33 (36%)	16/39 (41%)
Life Table Test	P=0.013	P=0.042	P=0.014
Incidental Tumour Test	P=0.009	P=0.098	P=0.019
Females
Adenoma
Overall	0/50 (0%)	0/50 (0%)	6/50 (12%)
Adjusted	0%	0%	17.4%
Terminal	0/15 (0%)	0/18 (0%)	3/30 (10%)
Life Table Test	P=0.012	(a)	P=0.067
Incidental Tumour Test	P=0.002	(a)	P=0.013
Carcinoma
Overall	1/50 (2%)	0/50 (0%)	4/50 (8%)
Adjusted	6.7%	0%	12.6%
Terminal	1/15 (7%)	0/18 (0%)	3/30 (10%)
Life Table Test	P=0.209	P=0.464N	P=0.401
Incidental Tumour Test	P=0.150	P=0.464N	P=0.302
Adenoma or Carcinoma ^(c)
Overall	1/50 (2%)	0/50 (0%)	10/50 (20%)
Adjusted	6.7%	0%	28.7%
Terminal	1/15 (7%)	0/18 (0%)	6/30 (20%)
Life Table Test	P=0.007	P=0.464N	P=0.050
Incidental Tumour Test	P=0.001	P=0.464N	P=0.008

^(a) No values are given because there was no tumour incidence in the dosed group or in the vehicle control group

^(b) Historical incidence at study laboratory (mean ± SD): 109/208 (37% ± 12%); historical incidence in NTP studies: 357/1091 (33% ± 10%)

^(c) Historical incidence at study laboratory (mean ± SD): 18/300 (6% ± 3%); historical incidence in NTP studies: 74/1092 (7% ± 4%)

Forestomach

Hyperplasia of the forestomach occurred at increased incidence in dosed animals of either sex. The incidences in dosed males and high-dose females were significantly higher than those of the controls. Squamous cell papillomas or carcinomas of the forestomach were increased in males. Squamous cell papillomas were also found in four high-dose females.

Table 4: Analysis of forestomach lesions in mice

	Vehicle control	500 mg/kg	1000 mg/kg
Males
Epithelial Hyerplasia	1/49 (2%)	7/48 (15%) ^(b)	22/49 (45%)^(b)
Squamous Cell Papilloma
Overall	3/49 (6%)	3/48 (6%)	9/49 (18%)
Adjusted	7.9%	9.1%	23.1%
Terminal	3/38 (8%)	3/33 (9%)	9/39 (23%
Life Table Test	P=0.038	P=0.597	P=0.065
Incidental Tumour Test	P=0.038	P=0.597	P=0.065
Squamous Cell Carcinoma	1/49 (2%)	1/48 (2%)	2/49 (4%)
Squamous Cell Papilloma or Carcinoma^(c)
Overall	4/49 (8%)	4/48 (8%)	11/49 (22%)
Adjusted	10%	11.3%	28.2%
Terminal	3/38 (8%)	3/33 (9%)	11/39 (28%)
Life Table Test	P=0.032	P=0.588	P=0.052
Incidental Tumour Test	P=0.028	P=0.619	P=0.051
Females
Epithelial Hyerplasia	1/49 (2%)	7/48 (15%) ^(b)	22/49 (45%) ^(b)
Squamous Cell Papilloma^(d)
Overall	0/50 (0%)	0/50 (0%)	4/48 (8%)
Adjusted	0%	0%	13.3%
Terminal	0/15 (0%)	0/18 (0%)	4/30 (13%)
Life Table Test	P=0.054	(a)	P=0.180
Incidental Tumour Test	P=0.054	(a)	P=0.180

^(a) No values are given because there was no tumour incidence in the dosed group or in the vehicle control group.

^(b) Significantly (P<0.05) higher than the controls

^(c) Historical incidence at study laboratory (mean ± SD); 2/296 (0.7% ± 2%); historical incidence in NTP studies: 14/1070 (1% ± 2%)

^(d) Historical incidence at study laboratory (mean ± SD): 2/297 (0.7% ± 1%); historical incidence in NTP studies: 3/1073 (0.3% ± 0.7%).

Multiple organs

Suppurative inflammation or abscesses of the ovaries, uterus, mesentry peritoneum or multiple organs were found in 26/35 (74%) controls, 14/32 (44%) low-dose and 8/20 (40%) high-dose females that died before termination.

Uterus/Endometrium

Cystic hyperplasia was found in 26/50 (52%) controls, 28/50 (56%) low-dose and 37/50 (74%) high-dose females.

Conclusions:

Under the conditions of the study, benzyl acetate increased the incidence of squamous cell neoplasms of the forestomach and hepatocellular adenomas. The method of dosing via gavage vehicle may have been a contributing factor. However reassessment of the conclusions of the results and conclusions as part of the 1993 feed study concluded that the lesions noted were due to the confounding influence of the corn oil vehicle and the route of exposure. Overall, from the combined reports, it was concluded that benzyl acetate did not demonstrate any evidence of carcinogenic activity under the conditions of this study.

Executive summary:

B6C3F1 mice (male and female) were administered with benzyl acetate via oral gavage in corn oil 5 days/week for 2 years at 500 and 1000 mg/kg bw/day. The methodology of the study was comparable to the OECD guideline 451; the differences between the methodology employed and the standardised guideline were not thought to influence the quality of the relevant results. The study was not performed to GLP, although the final date of the report is 1986, the in-life portion of the study pre-dated GLP inception. Under the conditions of the study benzyl acetate increased the incidence of squamous cell neoplasms and hepatocellular adenomas in both male and female mice. The confounding effect of the chosen method of dose delivery could not be separated from the effects recorded. A further study was performed in 1993, this time dosing the animals in feed rather than gavage, which allowed further assessment of the carcinogenic potential of benzyl acetate. From the reassessment of the results, with the confounding factors removed, it was concluded that benzyl acetate did not demonstrate any evidence of carcinogenic activity.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Study commenced 9 September 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid study is available for the analogue substance benzyl acetate. It is conducted in compliance with good scientific principles, with no or minor deviations from standard protocols. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Simonsen Laboratories, Inc. (Gilroy CA)- Age at study initiation: 40 days- Housing: Polycarbonate (Lab Products, Inc., Maywood NJ), changed twice weekly- Diet: Ad libitum- Water: Ad libitum- Acclimation period: 11 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22.6 °C ± 0.6 °C- Humidity (%): 45 to 52.6 %- Air changes (per hr): minimum of 10 changes/hour- Photoperiod (hrs dark / hrs light): 12 hours/day (fluorescent)IN-LIFE DATES: From: 25 August 1986 To: 23-25 November 1987 (interim sacrifice) and 22-26 August 1988 (terminal sacrifice)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION- Rate of preparation of diet (frequency): Weekly- Mixing appropriate amounts with (Type of food): NIH-07 open formula meal rat and mouse diet (Zeigler Brothers, Inc., Gardners, PA)- Storage temperature of food: -20 °C in the dark

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the (330 ppm) dose formulations were conducted. 10 g samples of dose formulations were extracted with 50 mL methanol and centrifuged, and 20 mL aliquots of the extracts were mixed with a 2 mL solution of valerophenone (0.66 mg/mL in methanol) and diluted to 25 mL with methanol. The solutions were analysed with gas chromatography with an FID and 10 % SP-2330 on 100/120 Supelcoport and a nitrogen carrier gas at a flow rate of 30 mL/minute with an oven temperature program of 120 °C for 20 minutes, then 120 °C to 200 °C at 10 °C/minute, with a 6-minute hold at 200 °C. Homogeneity was confirmed, stability (with losses of approximately 4 %) was established for at least 3 weeks when the dose formulations were stored in the dark at -20 °C. Dose formulations stored open to air and light showed significant loss (approximately 11 %).Based on the appearance of another peak during the gas chromatographic analysis, the dose formulations were analysed for benzyl alcohol. Benzyl alcohol was identified in the dose formulation by full mass scan gas chromatography/mass spectroscopy. A 10 g sample of the dose formulations was extracted with 50 mL methanol and analysed with a gas chromatographic system similar to that used in the homogeneity study but with a helium carrier gas and an oven temperature program of 40 °C for 3 minutes, then 40 °C to 225 °C at 10 °C/minute. For quantification of benzyl alcohol, dose formulations were extracted with methanol, 0.06 mg/mL valerophenone in methanol was added and the extracts were diluted further with methanol prior to gas chromatographic analysis. The system was the same as that used for the homogeneity studies, but the oven temperature was 110 °C. These analyses indicated concentrations of benzyl alcohol ranging from 0.9 % to 2.0 % relative to benzyl acetate concentrations after 3 days storage under simulated animal cage conditions.Dose formulations of benzyl acetate were analysed by the study laboratory using gas chromatography with the system described for the homogeneity study but with an oven temperature program of 125 °C for 5 minutes, then 125 °C to 140 °C at 5 °C/minute, with a 10-minute hold at 140 °C. Dose formulations were analysed every 6-8 weeks times during the study. All dose formulations were equal to or within 10 % of the target concentrations.

Duration of treatment / exposure:

2 years (up to 103 weeks)

Frequency of treatment:

Food was available ad libitum for during the course of the study and replenished daily.

Remarks:

Doses / Concentrations:0, 330, 1000 or 3000 ppmBasis:analytical conc.

No. of animals per sex per dose:

60

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The doses selected for this study were based primarily on lower body weight gains and lower final mean bodyweights of exposed mice in the 13 week study performed by the same laboratory.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: Twice dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: Clinical observations were recorded initially, weekly during the first 13 weeks of study, every 4 weeks thereafter, and at end of the study. BODY WEIGHT: Yes- Time schedule for examinations: Animal weights were recorded initially, weekly during the first 13 weeks of study, every 4 weeks thereafter, and at end of the study. 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: YesFeed consumption was measured daily per cage for 5 days once every 4 weeks.HAEMATOLOGY: Yes- Time schedule for collection of blood: Blood was collected from the retroorbital sinus at the 15 month interim evaluation.- Anaesthetic used for blood collection: No data- Animals fasted: No data- Parameters checked: Haematocrit, haemoglobin, erythrocytes, mean erythrocyte volume, mean erythrocyte haemoglobin,mean erythrocyte haemoglobin concentration, platelets, reticulocytes, and leucocyte count and differential.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Blood was collected from the retroorbital sinus at the 15 month interim evaluation.- Parameters checked: Cholesterol, triglyceride, alkaline phosphatase, creatine kinase and sorbitol dehydrogenase.URINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

HISTOPATHOLOGY: Yes.In addition to gross lesions, tissue masses and associated lymph nodes, tissue examination also included adrenal gland, brain oesophagus, femur (including marrow), heart, kidney, large intestine (cecum, colon and rectum), liver, lung, mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial or clitoral gland, prostate gland, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, tongue, trachea, urinary bladder and uterus.NECROPSYNecropsy was performed on all animals. Organs weighed were brain, right kidney and liver.

Other examinations:

A sentinel program was performed alongside the study investigations. The disease status of the rodents was monitored via serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals are subject to identical environmental conditions. The sentinel animals are from the same production source and weanling groups as the animals used for the study. During the 2 year study, 15 male and 15 female mice were selected at the time of randomisation and allocation of the animals to the various study groups. At 6, 12 and 18 months into the study, blood was drawn from five rats of each sex. Additional samples for viral screening at 24 months were collected from five male and five female control animals of each sex. Blood collected from each animals was allowed to clot and the serum separated. The following tests were performed:-ELISA:Ectromelia virus – 6, 12, 18 and 24 monthsCARB – 12 monthsGDVII – 6, 12, 18 and 24 monthsLCM (Lymphocytic choriomeningitis virus) – 6, 12 and 18 monthsM. arthritidis – 24 monthsM. pulmonis – 24 monthsMHV – 6, 12, 18 and 24 monthsMouse adenoma virus – 6, 12, 18 and 24 monthsMVM – 12, 18 and 24 monthsPVM – 6, 12, 18 and 24 monthsReovirus 3 – 6, 12, 18 and 24 monthsRCV/SDA – 6, 12, 18 and 24 monthsSendai – 6, 12, 18 and 24 months-Haemagglutination inhibition:K – 6, 12, 18 and 24 monthsMVM – 6 monthsPolyoma virus – 6, 12, 18 and 24 months-Immunofluorescent assay:EDIM – 6, 12, 18 and 24 monthsLCM – 24 monthsReo3 – 24 months

Statistics:

The results were analysed statistically examining the following: survival analyses, calculation of incidence, analysis of neoplasm incidences, analysis of nonneoplastic lesions incidences and analysis of continuous variables. Please refer to the field “Any other information on material and methods incl. tables” for a detailed description of the analyses performed in this study.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITYSurvival of exposed males was similar to that of the control groups. Survival of exposed females increased with exposure level with the survival of the 3000 ppm females significantly higher than that of the control group. Almost all deaths occurred during the last 9 months of the study; only 5 animals died during the first 15 months. No clinical findings were associated with the administration of benzyl acetate.BODY WEIGHT AND WEIGHT GAINMean bodyweights of exposed animals were consistently lower than those of the controls, except for the 330 ppm groups. Throughout the study, the mean bodyweight of 1000 ppm males was as much as 10% lower and that of the 3000 ppm males was as much as 13% lower than the control. Similarly the mean bodyweight of the 1000 ppm females was as much as 12% lower and that of the 3000 ppm females was as much as 16% lower than the control group throughout the study. The lower mean bodyweights of the 1000 and 3000 ppm males and females began after approximately 4 to 8 weeks of exposure and continued throughout the study. A decrease in mean bodyweight of all control and exposed males and females occurred between weeks 49 and 53 (males) and weeks 50 and 54 (females). The reason for this sudden decrease could not be determined. Recovery of the mean bodyweights was apparent by week 57 (males) and week 58 (females). FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)The average feed consumption by all exposed animals was similar to that of the control groups, however, feed consumption by 1000 and 3000 ppm males was lower that that of the control group at week 4. Dietary levels of 330, 1000 and 3000 ppm resulted in average daily consumptions of 35, 110 and 345 mg/kg bodyweight for males and 40, 130 and 375 mg/kg bodyweight for females.HAEMATOLOGYThe haematology profiles at the 15-month interim evaluations showed no test substance-related effects for the parameters measured.CLINICAL CHEMISTRYThe clinical chemistry results showed some differences in mean values for exposed groups compared to control groups. Triglycerides and cholesterol levels were slightly lower in exposed males and females and the alkaline phosphatase activity was lower in the 1000 and 3000 ppm females than in the control group.HISTOPATHOLOGY: NEOPLASTIC AND NON-NEOPLASTICNo increased incidences of neoplasms in males or females could be attributed to exposure to the test substance.The incidence of hepatocellular adenoma and carcinoma (combined) occurred with a dose related negative trend in males; the combined incidence in the 3000 ppm group was significantly lower than that in the control group (control, 22/50; 330 ppm, 22/520; 1000 ppm, 18/50; and 3000 ppm, 9/50). This effect did not occur in females.A significant decrease in the incidence of uterine stromal polyps occurred in 330 and 1000 ppm females (control, 8/50; 330 ppm, 2/49; 1000 ppm, 1/50; and 3000 ppm, 6/50).Dose-related increased incidences and severities of nonneoplastic lesions occurred in the nose of males and females of all exposure groups. The nasal lesions consisted of atrophy and degeneration, primarily of the olfactory epithelium, cystic hyperplasia of the nasal submucosal glands and exudate and pigmentation of the nasal mucosal epithelium. Atrophy consisted of depletion of bipolar neurons from the sensory cell layer of the olfactory epithelium, whereas degeneration was characterised by disorganisation of the remaining sensory and sustenacular cells with enlarged or occasionally pyknotic nuclei. The most commonly affected areas of the nose were the dorsal nasal septum, the junctional area (dorsal arch) of the dorsal septum and ethmoid turbinates and the dorsomedial aspect of the ethmoid turbinates. The lateral portion of the ethmoid turbinates as well as the entire nasal and maxillary turbinates were relatively less involved. The cells were interspersed with amorphous eosinophilic material, clear vacuoles, and occasionally, neutrophils. In some instances, the atrophied and degenerated epithelium contained gland-like structures several microns in diameter similar to hyperplastic Bowman's glands. The submucosal (Bowman's) glands in the affected regions were often hyperplastic and cystic and contained exudate in many animals. The epithelial cells of the hyperplastic glands were enlarged and occasionally extended upward to connect directly with the nasal meatus. Exudate was also present in the nasal meatus of some animals. A significant amount of finely granular, brown pigment was usually present in the affected olfactory epithelial cells and was similar to that in the olfactory epithelium of the controls; however, the amount of the pigment was increased. The pigment was not identified but was presumed to be an increase in the mucosal pigment normally seen in the olfactory epithelium. Occasionally, hyalinisation of the respiratory epithelium and the sustenacular cell layer of the olfactory epithelium was greater in severity that that observed in controls. The hyanalinization was characterised by the presence of abundant eosinophilic material, primarily in the goblet cells of the respiratory epithelium and in the sustenacular and mucus-producing cells of Bowman's glands in the olfactory epithelium (often referred to as hyaline degeneration).The nasal mucosal changes of atrophy, degeneration, and cystic hyperplasia of the glandular ducts occurred in the majority of male and female controls and the exposed animals. However, the severity of these lesions was greater and the incidence of the additional lesions of pigmentation was increased in the exposed animals in a dose-related manner. The atrophic and degenerative changes were usually minimal in severity, primarily in the anterior portions of the olfactory epithelium, and located dorsomedially in the control and 330 ppm groups progressed to moderate severity in the 3000 ppm males and females. The lesions in mice evaluated at the end of the study and those dying after 15 months were similar to those in mice evaluated at the interim evaluation, but were of greater severity. Whether the nasal changes resulted from a systemic effect of the ingested chemical, from a local effect of inhaled chemical vapour, or a combination of the mechanisms is unknown. No neoplasms or preneoplastic treatment-related lesions occurred in the nose.ANALOGUE APPROACH JUSTIFICATION:- See attached “Justification for read-across” document for full details.- In summary, important considerations for the use of read-across for acute toxicity are: i) 1-phenylethyl acetate (the target chemical) has similar physico-chemical properties as benzyl acetate (the source substance), ii) there are structural similarities between the two chemicals, iii) the OECD QSAR Toolbox assigns an identical toxicity profiles to both chemicals, and iv) both chemicals have been tested for acute oral toxicity, which demonstrated that neither substance requires classification for acute toxicity and that the benzyl acetate will represent a worst-case scenario, and are adequate for classification and labelling and risk assessment purposes. The information reported in this summary is included to demonstrate comparability between the source (benzyl acetate) and target (1-phenyl-ethyl acetate) substance.

Relevance of carcinogenic effects / potential:

The data presented in this study indicates no evidence of carcinogenic activity, contrary to the 1986 gavage study performed by the same laboratory. Within the discussions and conclusions of this study, the confounding factors of the previous study were discussed. Further data including genetic toxicity studies, toxicokinetics data, and other carcinogenicity data on the vehicle and test substance were assessed when drawing conclusions from the two studies. From the data available it was apparent that the test substance caused nonneoplastic nasal lesions with a dose-related response. The effects noted in the previous study were not evident in this study. A difference in dose levels could be construed as a contributing factor, however the available toxicokinetic data indicates that the effect may be related to the method of dosing (gavage versus feed), where the metabolic pathway is saturated due to the high temporary dose administered in gavage studies compared to the steady low level administration of the dose in feed over 24 hours.

Dose descriptor:

NOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Lesions were observed in the nose in all treatment groups. Mechanism of effects could not be determined. No neoplastic effects noted during the study.

Remarks on result:

other: Effect type: toxicity

Table 1: Survival of mice during the 2 year carcinogenicity feed study

Dose (ppm)	0	330	1000	3000
Male
Animals initially in study	60	60	60	60
15 month interim evaluation^a	10	10	10	10
Moribund	7	3	6	7
Natural deaths	4	4	3	4
Animals surviving to study termination	39	43	41	39
% probability of survival at end of study^b	78	86	82	78
Mean survival (days)	664	670	673	664
Survival analysis^c	P=0.718	P=0.444N	P=0.762N	P=0.840
Female
Animals initially in study	60	60	60	60
15 month interim evaluation^a	10	10	10	10
Moribund	12	16	7	3
Natural deaths	9	6	6	3
Animals surviving to study termination	29	29	37	44
% probability of survival at end of study^b	58	56	74	88
Mean survival (days)	644	640	667	663
Survival analysis^c	P=0.946	P=1.000N	P=0.134N	P=0.924

^aCensored from survival analysis

^bKaplan-Meier determinations

^cThe result of the life table trend test (Tarone, 1975) is in the control column and the results of the life table pairwise comparisons (Cox, 1972) with the controls are in the exposed columns. A negative trend of lower mortality in an exposure groups is indicated by N.

Table 2: Mean body weights and survival

	0 ppm		3000 ppm			6000 ppm			12,000 ppm
Weeks on study	Av. wt (g)	No. of survivors	Av. wt (g)	Wt (% of controls)	No. of survivors	Av. wt (g)	Wt (% of controls)	No. of survivors	Av. wt (g)	Wt (% of controls)	No. of survivors
Males
Terminal sacrifice		39			43			41			39
Mean for weeks
1-13	26.6		26.3	99		24.9	94		24.8	93
14-52	38.9		38.2	98		36.5	94		36.1	93
53-101	44.1		43.3	98		41.0	93		39.7	90
Females
Terminal sacrifice		29			28			37			44
Mean for weeks
1-13	23.4		22.6	97		21.3	91		20.6	88
14-52	35.6		34.8	98		32.3	91		31.6	89
53-101	42.0		42.0	100		37.7	90		36.9	88

Table 3: Incidences of nonneoplastic lesions of the nose^a

Dose (ppm)	0	330	1000	3000
Male
15 month interim evaluation
n	10	10	10	10
Mucosa, atrophy	0	8** (0.8)**	10** (1.8)**	10** (2.6)**
Mucosa, degeneration	5 (0.5)	5 (0.6)	10* (1.9)**	10** (2.7)**
Glands, cystic hyerplasia	2 (0.2)	3 (0.3)	1 (0.1)	10** (1.3)**
Exudate	0	0	1	5*
Mucosa, pigmentation	0	7** (0.7)**	10** (1.3)**	10** (2.0)**
2 year study
n	50	50	50	50
Mucosa, atrophy	30 (0.6)	49** (1.0)**	50** (2.0)**	50** (2.9)**
Mucosa, degeneration	31 (0.6)	50** (1.0)**	50** (2.0)**	50** (2.9)**
Glands, cystic hyperplasia	22 (0.7)	43** (1.1)**	47** (1.2)**	50** (1.9)**
Exudate	8	18*	38**	26**
Mucosa, pigmentation	0	45** (0.9)**	50** (1.2)**	50** (2.0)**
Female
15 month interim evaluation
n	10	10	10
Mucosa, atrophy	5 (0.5)	9 (0.9)	10* (2.2)**	10* (3.0)**
Mucosa, degeneration	10 (1.1)	9 (0.9)	10 (2.2)**	10 (3.0)**
Glands, cystic hyperplasia	8 (0.8)	9 (0.9)	10 (1.1)	10 (2.1)**
Exudate	0	0	0	2
Mucosa, pigmentation	0	7** (0.7)**	10** (1.4)**	10** (1.8)**
2 year study
n	50	50	50	50
Mucosa, atrophy	41 (0.8)	48* (1.2)**	49** (2.4)**	50** (2.8)**
Mucosa, degeneration	48 (1.0)	48 (1.2)*	0 (2.3)**	50 (2.8)**
Glands, cystic hyperplasia	39 (1.3)	45 (1.1)	49** (1.4)**	50** (1.9)**
Exudate	15	26*	36**	43**
Mucosa, pigmentation	0	46** (0.9)**	48** (1.3)**	48** (1.5)**

*significantly different (P≤0.05) from the control group by Fisher's exact test 915 month interim) or logistic regression (2 year)

**P≤0.01

^aanimals examined microscopically and number of animals with lesion; average severity grade (in parentheses) for all animals: 0=none, 1=minimal, 2=mild, 3=moderate, 4=marked

Conclusions:

Under the conditions of the study, there was no evidence of carcinogenic activity of the test substance in male or female mice receiving 330, 1000 or 3000 ppm.

Executive summary:

Groups of 60 male and 60 female B6C3F1 mice were fed diets containing 0, 330, 1000 or 3000 ppm benzyl acetate for 2 years. Survival of exposed mice was similar to that of the controls except in 3000 ppm females. In this group survival was found to be significantly higher than the control group. Throughout the study, the mean bodyweights of 1000 and 3000 ppm males and females were 2% to 14% lower than those of the control groups. Dietary levels of 330, 1000 and 3000 ppm benzyl acetate was estimated to result in average daily consumption levels of 35, 110, and 345 mg/kg (males) and 40, 130 and 375 mg/kg (females). No biologically significant changes in haematology or clinical chemistry parameters were observed in mice receiving 330, 1000 or 3000 ppm benzyl acetate. No increase in neoplasm incidence in mice could be attributed to benzyl acetate administration in feed. Dose-related increased incidences or severities of nonneoplastic nasal lesions occurred in the most posterior portions of the nasal cavity in all exposed groups. The lesions occurred in the majority of the exposed mice and consisted of atrophy and degeneration, primarily of the olfactory epithelium, cystic hyperplasia of the nasal submucosal glands, pigmentation of the mucosal epithelium, and exudate accumulation.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

Study commenced November 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid study is available for the analogue substance benzyl acetate. It is conducted in compliance with good scientific principles, with no or minor deviations from standard protocols. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Principles of method if other than guideline:

The general principles of the methodology are similar to method OECD 451, minor deviations are apparent, such as dosing five days per week instead of seven, which the guideline states may be acceptable and only two dosing levels rather than the recommended three were employed.

GLP compliance:

not specified

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Breeding Laboratories, Portage, MI- Age at study initiation: 7 weeks- Housing: Polycarbonate, Lab Products Inc, Garfield, NJ, USA- Diet (e.g. ad libitum): Wayne Lab-Blox, Allied Mills Inc, Chicago ad libitum- Water (e.g. ad libitum): tap water ad libitum- Acclimation period: 3 weeksENVIRONMENTAL CONDITIONS- Temperature (°C): 21-24 ºC- Humidity (%): 30-60%- Air changes (per hr): 15 per hour- Photoperiod (hrs dark / hrs light): 12 hours of fluorescent light per day

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS- Doses were prepared on a weight-to-volume basis by pipetting the appropriate amount of benzyl acetate into a vessel and adding enough corn oil to give the desired concentration. Solutions were mixed until they were visually homogenous. Once prepared, mixtures were stored at 5 °C for no more than 11 days.VEHICLE- Justification for use and choice of vehicle (if other than water): Although exposure to benzyl acetate is primarily in food, the study opted to dose in corn oil via gavage due to the substance’s volatility and it’s reactivity with moisture present in feed.- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of benzyl acetate in corn oil were selected at random and analysed periodically at Southern Research Institute.Method used until 04.04.79:Samples of benzyl acetate were received as corn oil mixtures. 0.5 ml aliquots were dissolved in 10 ml chloroform. Analysis was by vapour-phase chromatography under the following conditions:Instrument: Perkin-Elmer 910Detection: Flame ionizationColumn: 3% OV-17 on 80/100 Suplecoport, 1.8 m x 4 mm I.D., glassTemperatures: Inlet, 140 ºC; Oven, 100 ºC, isothermal; Detector, 170 ºCInjection Size: 2 µlRetention time: 2.8 minThere was no correction for workup loss since samples were injected without any extraction or workup procedure. The gavage samples were compared with the reference standards of benzyl acetate prepared volume/volume in corn oil, dissolved in chloroform in the same manner as the gavage samples and analysed under the same chromatographic conditions.Method used after 04.04.79:Samples of benzyl acetate were received as corn oil mixtures in sealed syringe bottles. Samples were extracted with methanol for 3 minutes (20 ml methanol with 0.5ml of sample made up in corn oil). Analysis was by vapour-phase chromatography under the following conditions:Instrument: Sigma 1Detection: Flame ionizationColumn: 3% OV-17 on 80/100 Suplecoport, 1.8 m x 4 mm I.D., glassTemperatures: Inlet, 140 ºC; Oven, 100 ºC, isothermal; Detector, 170 ºCCarrier gas: HeliumInjection Size: 1 µlRetention time: 2.5 minThe gavage samples were compared with the reference standards of benzyl acetate prepared volume/volume in corn oil then extracted with methanol in the same manner as the sample. There was no correction applied to the samples since samples and reference standard were treated in the same manner.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

5 days per week

Post exposure period:

Not applicable, terminal kill was performed at 104-106 weeks.

Remarks:

Doses / Concentrations:0, 250 and 500 mg/kg bwBasis:

No. of animals per sex per dose:

Fifty

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: on the basis of the results from the 13 week study

Observations and examinations performed and frequency:

MORTALITY AND MORBIDITY: Yes- Time schedule: twice daily. Animals that were judged to be moribund were killed with carbon dioxide and necropsied.DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: at each weighing period - weekly for first 13 weeks, monthly until week 91 and then every two weeksBODY WEIGHT: Yes- Time schedule for examinations: every week for the first 13 weeks and monthly thereafterFOOD 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: NoWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): NoOPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: NoCLINICAL CHEMISTRY: NoURINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: YesNecropsies were performed on all animals found dead and on those killed at the end of the study unless precluded in whole or in part by autolysis or cannibalisation.HISTOPATHOLOGY: YesThe following organs were examined microscopically gross lesions, brain, pituitary, thymus, spleen, thyroid, parathyroid, lung and bronchi, trachea, heart, oesphagus, stomach (pylorus and fundus), duodenum, jejunum, ileum, large intestine, pancreas, adrenal, kidney, liver, skin, mammary gland, urinary bladder, prostate, seminal vesicles, testes or uterus/ovaries, femur with marrow, abnormal lymph nodes, salivary gland, thigh muscle, sciatic nerve, costochondral junction, larynx, mesenteric lymph nodes, nasal cavity and spinal cord

Other examinations:

Animal disease condition at the laboratory was monitored by the use of sentinel animals. Plasma samples were obtained from these animals (5 per sex and species) at 6, 12, and 18 months and from vehicle control animals at 24 months. These samples were assayed for the various viral serology titers.

Statistics:

SURVIVAL ANALYSISThe probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented graphically. Animals were censored from survival analysis at the time they were found dead of other than natural causes. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups of equality and Tarone's (1975) extensions of Cox's method for testing a dose-related trend. All reported P values are two-sided.CALCULATION OF INCIDENCE RATESThe incidence of neoplastic or non-neoplastic lesions has been given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which the site was examined. In most instances, the denominators include those animals for which the site was examined histologically. However, when macroscopic examination was required to detect lesions prior to histologic sampling, or when lesions could have appeared at multiple sites, the denominators consist of the number of animals on which necropsies were performed.HISTORICAL CONTROL DATAAlthough the concurrent control group is always the first and most appropriate control group used for decision-making, there are certain instances in which historical control data can be helpful in the overall evaluation of tumour incidence. Consequently, control tumour incidence from the NTP historical control database (Haseman et al., 1984) are included for those tumours in these studies appearing to show substance-related effects.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITYNo significant differences in survival were observed between any groups of either sex. In males, 38/50 (76%) of the controls, 46/50 (92%) of the low-dose group and 40/50 (80%) of the high-dose group lived to the termination period of the study. In females, 40/50 (80%) of the controls, 36/50 (72%) of the low-dose group and 36/50 (72%) of the high-dose group lived to the termination period of the study. These results include one control male, one low-dose male, one control female and one low-dose female which died during the termination period of the study and are considered to have been killed at the end of the study.BODY WEIGHT AND WEIGHT GAINMean bodyweights of dosed and control male rats were comparable throughout most of the study (see table 1). After week 20, mean bodyweights of the high-dose females were slightly greater than those of the controls.HISTOPATHOLOGYAcinar-cell adenomas in the pancreas of males occurred with a positive trend and the incidence of in the high-dose group was significantly higher than in the vehicle controls. The incidence of these tumours in the low-dose group was comparable to that in the gavage controls. Acinar-cell hyperplasia of the pancreas was observed in control, low-dose and high-dose males. No acinar-cell hyperplasia of the pancreas was observed in females.The incidence of retinopathy and cataracts in the high-dose males was increased compared with the controls. Low-dose females had an increased incidence of retinopathy. These effects were associated with proximity to fluorescent lighting.Preputial gland neoplasms occurred with a positive trend in males (cystadenocarcinoma, all adenocarcinoma and adenocarcinoma and carcinoma combined). However the incidence of all preputial gland tumours was not significantly elevated. For females the incidence of clitoral gland neoplasms was marginally increased.ANALOGUE APPROACH JUSTIFICATION:- See attached “Justification for read-across” document for full details.- In summary, important considerations for the use of read-across for acute toxicity are: i) 1-phenylethyl acetate (the target chemical) has similar physico-chemical properties as benzyl acetate (the source substance), ii) there are structural similarities between the two chemicals, iii) the OECD QSAR Toolbox assigns an identical toxicity profiles to both chemicals, and iv) both chemicals have been tested for acute oral toxicity, which demonstrated that neither substance requires classification for acute toxicity and that the benzyl acetate will represent a worst-case scenario, and are adequate for classification and labelling and risk assessment purposes. The information reported in this summary is included to demonstrate comparability between the source (benzyl acetate) and target (1-phenyl-ethyl acetate) substance.

Relevance of carcinogenic effects / potential:

The data presented in this study indicates some carcinogenic potential based on the results seen in the males, however the report was reviewed many times, and the results divided the reviewing panel. The reviews questioned the dosing methods and the impact on the results on multiple occasions. A further two year study was performed in 1993 by the same group, this time dosing in feed rather than via gavage.When assessing the second study a larger collection of genetic toxicity studies, toxicokinetics data, and other carcinogenicity data on the vehicle and test substance was available. From the wealth of available data it was demonstrated that the administration of corn oil is associated with the observed effects, and benzyl acetate is a weak promoter. The overwhelming conclusions indicated that the dosing method in this report did confound the results, and benzyl acetate did not demonstrate any carcinogenicity.

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Acinar cell adenomas observed in both dose groups tested.

Remarks on result:

not determinable

Remarks:

no NOAEL identified. Effect type:carcinogenicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No treatment related lesions reported

Remarks on result:

other: Effect type: carcinogenicity )

Table 1: Mean bodyweights and survival of rats

	Control		250 mg/kg			500 mg/kg
Weeks on study	Average wt (g)	No. of survivors	Average wt (g)	Wt (% of controls)	No. of survivors	Average wt (g)	Wt (%) of controls	No. of survivors
Male
0	133	50	133	100	50	133	100	50
1	185	50	181	98	50	179	97	50
2	209	50	207	99	50	203	97	50
3	229	50	219	96	50	219	96	50
4	244	50	238	98	50	230	94	50
5	261	50	256	98	50	249	95	50
6	276	50	270	98	50	263	95	50
7	289	50	281	97	50	275	95	50
8	298	50	290	97	50	285	96	50
9	308	50	300	97	50	293	95	50
10	317	50	310	98	50	302	95	50
11	328	50	319	97	50	311	95	50
12	333	50	325	98	50	317	95	50
13	338	50	331	98	50	322	95	50
18	358	50	352	98	50	344	96	50
22	377	50	369	98	50	362	96	50
27	389	50	378	97	50	368	95	50
31	398	50	388	97	50	382	96	50
36	421	50	408	97	50	400	95	50
40	428	50	416	97	50	409	96	50
44	431	50	420	97	50	411	95	50
48	432	50	421	97	50	417	97	50
53	447	50	436	98	50	431	96	50
58	461	50	455	99	50	447	97	50
62	467	50	453	97	50	445	95	49
66	473	50	459	97	50	456	96	49
70	478	50	467	98	50	463	97	49
74	481	50	472	98	50	476	99	49
80	485	49	476	98	50	482	99	46
84	481	48	478	99	48	488	101	46
89	475	46	476	100	48	487	103	45
93	473	41	475	100	47	488	103	45
96	465	41	474	102	47	488	105	44
101	456	40	470	103	47	489	107	43
104	448	38	447	100	46	484	108	40
Female
0	106	50	107	101	50	107	101	50
1	129	50	129	100	50	129	100	50
2	139	50	137	99	50	139	100	50
3	145	50	144	99	50	144	99	50
4	154	50	152	99	50	151	98	50
5	160	50	160	100	50	162	101	50
6	165	50	165	100	50	166	101	50
7	171	50	171	100	50	172	101	49
8	175	50	175	100	50	176	101	49
9	178	50	177	99	50	178	100	49
10	181	50	181	100	50	182	101	49
11	185	50	183	99	50	186	101	49
12	188	50	186	99	50	186	99	49
13	188	50	187	99	50	190	101	49
18	194	50	194	100	50	197	102	49
22	199	50	201	101	50	204	103	49
27	203	50	204	100	50	208	102	49
31	208	50	206	99	50	213	102	49
36	215	50	216	100	50	224	104	49
40	220	50	217	99	50	225	102	49
44	225	50	222	99	50	229	102	49
48	224	50	225	100	50	233	104	49
53	229	50	234	102	50	239	104	49
58	238	50	243	102	50	249	105	49
62	243	50	244	100	50	245	101	49
66	249	50	251	101	50	257	103	49
70	258	49	261	101	50	269	104	49
74	264	49	268	102	48	277	105	49
80	273	48	275	101	47	289	106	47
84	275	48	278	101	44	297	108	47
89	276	48	280	101	43	293	106	43
93	279	46	282	101	42	295	106	43
96	278	46	285	103	39	294	106	42
101	280	42	282	101	38	300	107	39
104	279	40	282	101	36	297	106	36

Table 2: Survival of rats

	Vehicle control	250 mg/kg	500 mg/kg
Male
Animals initially in study	50	50	50
Natural death or moribund sacrifice	12	4	9
Accidental death or missing	0	0	1
Scheduled or terminal sacrifice	38	46	40
Survival P values ^(a)	0.522	0.058	0.638
Female
Animals initially in study	50	50	50
Natural death or moribund sacrifice	10	13	14
Accidental death or missing	0	1	0
Scheduled or terminal sacrifice	40	36	36
Survival P values ^(a)	0.394	0.534	0.442

^(a) The vehicle control column contains the result of the life table trend test; the columns for dosed groups contain the life table pairwise comparisons with the vehicle controls.

Pathology and Statistical Analyses of Results

Pancreas

Acinar-cell hyperplasia was observed in all groups of males. While the total number of animals with hyperplasia was fairly constant across dose groups, the hyperplastic lesions were more frequently multiple in the treated animals. Acinar-cell hyperplasia can be recognised as a focal area that is more eosinophilic and stains more intensely than the adjacent pancreas. Hyperplastic lesions have cytological features similar to adenomas such as variation in cell size, nuclear pleomorphism and increased miotic activity. However, lesions diagnosed as hyperplasias are smaller than adenomas, show little or no compression and often appear contiguous with the adjacent unaffected parenchyma. Acinar cell adenomas in male rats occurred with a statistically significant positive trend. Results of the pairwise comparison with controls were significant for the high-dose group. The lesion was more often multiple in the treated animals. This tumour was not observed in any females. The cells in the pancreatic adenomas contained zymogen granules and were arranged like normal acinar cells except that there was a lack of islet cells. Cells in the pancreatic lesions had slightly larger nuclei and the miotic index was increased. Because of the increased miotic activity and mild compression of adjacent tissues that caused a distinct line of demarcation, the pancreatic lesions were classified as acinar-cell adenomas.

Table 3: Analysis of acinar-cell adenoma of the pancreas in male rats

	Vehicle control	250 mg/kg	500 mg/kg
Hyperplasia	37/50	34/50	36/49
Hyperplasia, multifocal	15/50	18/50	25/49
Adenoma, single ^(a)	12/50 (24%)	15/50 (30%)	15/49 (31%)
Adenoma, multiple ^(a)	10/50 (20%)	12/50 (24%)	22/49 (45%)
Overall	22/50 (44%)	27/50 (54%)	37/49 (76%)
Adjusted	53.6%	58.7%	82.2%
Terminal	19/38 (50%)	27/46 (59%)	32/40 (80%)
Life Table Test	P=0.003	P=0.548	P=0.007
Incidental Tumor Test	P=0.001	P=0.353	P=0.001

^(a) Historical incidence of acinar-cell adenoma or carcinomas (combined) at study laboratory (mean ± SD): 14/298 (5% ± 9%); historical incidence in NTP studies: 47/1086 (4% ± 7%).

Subcutaneous tissue

The incidence of low-dose males with subcutaneous fibromas was significantly higher than that of controls. Trend test results and comparisons between high-dose group and controls were not significant. The incidences of this tumour in males were: control 3/50, 6%; low-dose 11/50, 22%, high-dose 5/50, 10%.

Preputial gland

Tumours observed with statistically significant positive trends in males include cystadenocarcinoma, all adenocarcinoma and adenocarcinoma or carcinoma combined. Results of the pairwise comparisons with the controls were not statistically significant and the combined incidence of adenomas, adenocarcinomas or carcinomas were not statistically significant. In females, clitoral tumours did not occur in statistically significant proportions: control 2/50, 4%; low-dose 0/50; high-dose 5/50, 10%.

Table 4: Analysis of preputial gland tumours in male rats

	Vehicle control	250 mg/kg	500 mg/kg
Adenoma	1/50	0/50	0/50
Cystadenocarcinoma
Overall	0/50 (0%)	0/50 (0%)	3/50 (6%)
Adjusted	0%	0%	7.5%
Terminal	0/38 (0%)	0/46 (0%)	3/40 (8%)
Life Table Test	P=0.036	(a)	P=0.130
Incidental Tumour Test	P=0.036	(a)	P=0.130
Adenocarcinoma
Overall	0/50 (0%)	1/50 (2%)	4/50 (8%)
Adjusted	0%	2.2%	10%
Terminal	0/38 (0%)	1/46 (2%)	4/40 (10%)
Life Table Test	P=0.025	P=0.538	P=0.070
Incidental Tumour Test	P=0.025	P=0.538	P=0.070
Carcinoma	1/50	0/50	2/50
Adenocarcinoma or Carcinoma
Overall	1/50 (2%)	1/50 (2%)	6/50 (12%)
Adjusted	2.6%	2.2%	14.3%
Terminal	1/38 (3%)	1/46 (2%)	5/40 (13%)
Life Table Test	P=0.023	P=0.719N	P=0.067
Incidental Tumor Test	P=0.040	P=0.719N	P=0.092
Adenoma, Adenocarcinoma or Carcinoma ^(b)
Overall	2/50 (4%)	1/50 (2%)	6/50 (12%)
Adjusted	5.3%	2.2%	14.3%
Terminal	2/38 (5%)	1/46 (2%)	5/40 (13%)
Life Table Test	P=0.073	P=0.433N	P=0.150
Incidental Tumour Test	P=0.110	P=0.433N	P=0.194

^(a) No values are given because there was no tumour incidence in the dosed group or in the vehicle control group.

^(b) Historical incidence at study laboratory (mean ± SD); 11/300 (4% ± 3%); historical incidence in NTP studies: 42/1100 (4% ± 4%)

Eye

Increased incidences of retinopathy and cataracts were observed in high-dose males: controls 1/50, 2%; low-dose 0/50; high-dose 20/50, 40% (retinopathy) and controls 0/50; low-dose 0/50; high-dose 13/50, 26% (cataracts). In females the following incidences were observed: control 0/50; low-dose 18/50, 36%; high-dose 1/50, 2% (retinopathy) and controls 0/50; low-dose 3/50, 6%; high-dose 0/50 (cataracts).

Testis

Interstital-cell tumours occurred with a statistically significant negative trend: control 48/50, 96%; low-dose 48/50, 96%; high-dose 37/50, 74%. The incidence in the high-dose group was statistically significant in pairwise comparisons with the controls. There was a significant increase in the incidence of high-dose males with interstitial-cell hyperplasia: control 4/50, 8%; low-dose 2/50, 4%; high-dose 17/50, 34%.

Conclusions:

Under the conditions of the test, the test substance increased the incidence of acinar-cell adenomas of the exocrine pancreas in males. The gavage vehicle may have been a contributing factor. No evidence of carcinogenicity was found for females. However reassessment of the conclusions of the results and conclusions as part of the 1993 feed study concluded that the lesions noted were due to the confounding influence of the corn oil vehicle and the route of exposure. Overall, from the combined reports, it was concluded that the test substance did not demonstrate any evidence of carcinogenic activity.

Executive summary:

Fischer 344 rats (male and female) were administered with benzyl acetate via oral gavage in corn oil 5 days/week for 2 years. The methodology of the study was comparable to the OECD guideline 451; the differences between the methodology employed and the standardised guideline were not thought to influence the quality of the relevant results. The study was not performed to GLP, although the final date of the report is 1986, the in-life portion of the study pre-dated GLP inception. Under the conditions of the study benzyl acetate increased the incidence of acinar-cell adenomas of the exocrine pancreas in males. No evidence of carcinogenicity was found for females. The confounding effect of the corn oil vehicle and the chosen method of dose delivery could not be separated from the effects recorded. A further study was performed in 1993, this time dosing the animals in feed rather than gavage, which allowed further assessment of the carcinogenic potential of benzyl acetate. From the reassessment of the results, with the confounding factors removed, it was concluded that benzyl acetate did not demonstrate any evidence of carcinogenic activity.

Reference 4

Endpoint:

carcinogenicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Study commenced 9 September 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

TA valid study is available for the analogue substance benzyl acetate. It is conducted in compliance with good scientific principles, with no or minor deviations from standard protocols. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Simonsen Laboratories, Inc. (Gilroy CA)- Age at study initiation: 41 days- Housing: Polycarbonate (Lab Products, Inc., Maywood NJ), changed twice weekly- Diet: Ad libitum- Water: Ad libitum- Acclimation period: 12 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 21.9 °C ± 0.4 °C- Humidity (%): 45.4 to 54 %- Air changes (per hr): minimum of 10 changes/hour- Photoperiod (hrs dark / hrs light): 12 hours/day (fluorescent)IN-LIFE DATES: From: 9 September 1986 To: 8-10 December 1987 (interim sacrifice) and 6-12 September 1988 (terminal sacrifice)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION- Rate of preparation of diet (frequency): Weekly- Mixing appropriate amounts with (Type of food): NIH-07 open formula meal rat and mouse diet (Zeigler Brothers, Inc., Gardners, PA)- Storage temperature of food: -20 °C in the dark

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the (330 ppm from the mouse study) dose formulations were conducted. 10 g samples of dose formulations were extracted with 50 mL methanol and centrifuged, and 20 mL aliquots of the extracts were mixed with a 2 mL solution of valerophenone (0.66 mg/mL in methanol) and diluted to 25 mL with methanol. The solutions were analysed with gas chromatography with an FID and 10 % SP-2330 on 100/120 Supelcoport and a nitrogen carrier gas at a flow rate of 30 mL/minute with an oven temperature program of 120 °C for 20 minutes, then 120 °C to 200 °C at 10 °C/minute, with a 6-minute hold at 200 °C. Homogeneity was confirmed, stability (with losses of approximately 4 %) was established for at least 3 weeks when the dose formulations were stored in the dark at -20 °C. Dose formulations stored open to air and light showed significant loss (approximately 11 %).Based on the appearance of another peak during the gas chromatographic analysis, the dose formulations were analysed for benzyl alcohol. Benzyl alcohol was identified in the dose formulation by full mass scan gas chromatography/mass spectroscopy. A 10 g sample of the dose formulations was extracted with 50 mL methanol and analysed with a gas chromatographic system similar to that used in the homogeneity study but with a helium carrier gas and an oven temperature program of 40 °C for 3 minutes, then 40 °C to 225 °C at 10 °C/minute. For quantification of benzyl alcohol, dose formulations were extracted with methanol, 0.06 mg/mL valerophenone in methanol was added and the extracts were diluted further with methanol prior to gas chromatographic analysis. The system was the same as that used for the homogeneity studies, but the oven temperature was 110 °C. These analyses indicated concentrations of benzyl alcohol ranging from 0.9 % to 2.0 % relative to benzyl acetate concentrations after 3 days storage under simulated animal cage conditions.Dose formulations of benzyl acetate were analysed by the study laboratory using gas chromatography with the system described for the homogeneity study but with an oven temperature program of 125 °C for 5 minutes, then 125 °C to 140 °C at 5 °C/minute, with a 10-minute hold at 140 °C. Dose formulations were analysed every 6-8 weeks times during the study. All dose formulations were equal to or within 10 % of the target concentrations.

Duration of treatment / exposure:

2 years (up to 103 weeks)

Frequency of treatment:

Food was available ad libitum for during the course of the study and replenished daily.

Remarks:

Doses / Concentrations:0, 3000, 6000 or 12000 ppmBasis:analytical conc.

No. of animals per sex per dose:

60

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The doses selected for this study were based on lower survival, mean body weights and feed consumption and on increased incidences of histopatholigic brain lesions in 50000 ppm male and female rats in the 13 week study of similar design.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: Twice dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: Clinical observations were recorded initially, weekly during the first 13 weeks of study, every 4 weeks thereafter, and at end of the study. BODY WEIGHT: Yes- Time schedule for examinations: Animal weights were recorded initially, weekly during the first 13 weeks of study, every 4 weeks thereafter, and at end of the study. 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: YesFeed consumption was measured daily per cage for 5 days once every 4 weeks.HAEMATOLOGY: Yes- Time schedule for collection of blood: Blood was collected from the retroorbital sinus at the 15 month interim evaluation. Pancreas samples (interim evaluation male rats) were also collected.- Anaesthetic used for blood collection: No data- Animals fasted: No data- Parameters checked: Haematocrit, haemoglobin, erythrocytes, mean erythrocyte volume, mean erythrocyte haemoglobin,mean erythrocyte haemoglobin concentration, platelets, reticulocytes, and leucocyte count and differential.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Blood was collected from the retroorbital sinus at the 15 month interim evaluation. Pancreas samples (interim evaluation male rats) were also collected.- Anaesthetic used for blood collection: No data- Animals fasted: No data- Parameters checked: Cholesterol, triglyceride, alkaline phosphatase, creatine kinase and sorbitol dehydrogenase. Pancreatic enzymes amylase, lipase and carboxypeptidase.URINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

HISTOPATHOLOGY: Yes.In addition to gross lesions, tissue masses and associated lymph nodes, tissue examination also included adrenal gland, brain oesophagus, femur (including marrow), heart, kidney, large intestine (cecum, colon and rectum), liver, lung, mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial or clitoral gland, prostate gland, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, tongue, trachea, urinary bladder and uterus.NECROPSYNecropsy was performed on all animals. Organs weighed were brain, right kidney and liver.

Other examinations:

A sentinel program was performed alongside the study investigations. The disease status of the rodents was monitored via serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals are subject to identical environmental conditions. The sentinel animals are from the same production source and weanling groups as the animals used for the study. During the 2 year study, 15 male and 15 female rats were selected at the time of randomisation and allocation of the animals to the various study groups. At 6, 12 and 18 months into the study, blood was drawn from five rats of each sex. Additional samples for viral screening at 24 months were collected from five male and five female control animals of each sex. Blood collected from each animals was allowed to clot and the serum separated. The following tests were performed:- ELISA:CARB (cilia-associated respiratory bacillus) – 12 monthsM. arthritidis – 24 monthsM. pulmonis – 24 monthsPVM – 6, 12, 18 and 24 monthsRCV/SDA – 6, 12, 18 and 24 monthsSendai – 6, 12, 18 and 24 months- Haemagglutination inhibition:H-1 – 6, 12, 18 and 24 monthsKRV – 6, 12, 18 and 24 months

Statistics:

The results were analysed statistically examining the following: survival analyses, calculation of incidence, analysis of neoplasm incidences, analysis of nonneoplastic lesions incidences and analysis of continuous variables. Please refer to the field “Any other information on material and methods incl. tables” for a detailed description of the analyses performed in this study.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITYThe survival rates among the exposed animals were similar to those of the control. No clinical findings were associated with the administration of the test substance.BODY WEIGHT AND WEIGHT GAINThe mean body weight of males receiving 3000 and 6000 ppm were similar to those of the control group throughout the study. The mean bodyweights of the 12000 ppm males and exposed females were approximately 5 % lower than those of the control groups throughout most of the study.FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)The average feed consumption by all exposure groups was similar to that of the control groups, with only the 12000 ppm males consuming slightly less feed throughout the study. Dietary levels of 3000, 6000 and 12000 ppm resulted in average daily consumption of 130, 260 and 510 mg/kg for males and 145, 290 and 575 mg/kg for females respectively.HAEMATOLOGYThe haematology profiles of animals at the 15-month interim evaluations showed no test substance-related effects for the parameters measured.CLINICAL CHEMISTRYAlkaline phosphatase activity was slightly higher in 12,000 ppm males than in control males, but the relationship to test substance administration was inconclusive. Pancreatic enzyme activities were measured in males only, but no significant differences occurred between any exposure group and the control groups for any of the enzymes.HISTOPATHOLOGY: NEOPLASTIC AND NON-NEOPLASTICNo biologically noteworthy changes in the incidences of neoplasms or nonneoplastic lesions occurred in any organ in males or females, except for a significant decrease in the incidence on uterine stromal polyps in the 6000 ppm females (control 15/60; 3000 ppm 18/60, 6000 ppm 4/60 and 12,000 ppm 11/60) which was not considered related to treatment. HISTORICAL CONTROL DATAThe incidences of various lesions in all rat groups were within the normal range for F344/N rats in these type of studies.ANALOGUE APPROACH JUSTIFICATION:- See attached “Justification for read-across” document for full details.- In summary, important considerations for the use of read-across for acute toxicity are: i) 1-phenylethyl acetate (the target chemical) has similar physico-chemical properties as benzyl acetate (the source substance), ii) there are structural similarities between the two chemicals, iii) the OECD QSAR Toolbox assigns an identical toxicity profiles to both chemicals, and iv) both chemicals have been tested for acute oral toxicity, which demonstrated that neither substance requires classification for acute toxicity and that the benzyl acetate will represent a worst-case scenario, and are adequate for classification and labelling and risk assessment purposes. The information reported in this summary is included to demonstrate comparability between the source (benzyl acetate) and target (1-phenyl-ethyl acetate) substance.

Relevance of carcinogenic effects / potential:

The data presented in this study indicates no evidence of carcinogenic activity, contrary to the 1986 gavage study performed by the same laboratory. Within the discussions and conclusions of this study, the confounding factors of the previous study were discussed. Further data including genetic toxicity studies, toxicokinetics data, and other carcinogenicity data on the vehicle and test substance were assessed when drawing conclusions from the two studies. From the data available it was apparent that test substance is a weak promoter and the effects noted in the previous study were due to the method of dosing and the vehicle.

Dose descriptor:

NOAEL

Effect level:

12 000 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Effect type: carcinogenicity

Remarks:

12000ppm = 510 mg/kg/bw (male) / 570 mg/kg bw (female)

Table 1: Survival during the 2 year carcinogenicity feed study

Dose (ppm)	0	3000	6000	12000
Male
Animals initially in study	60	60	60	60
15 month interim evaluation^a	10	10	10	10
Moribund	20	12	16	14
Natural deaths	3	4	4	3
Animals surviving to study termination	27	34	30	33
% probability of survival at end of study^b	54	68	60	66
Mean survival (days)	646	49	658	652
Survival analysis^c	P=0.455N	P=0.291N	P=0.632N	P=0.346N
Female
Animals initially in study	60	60	60	60
15 month interim evaluation^a	10	10	10	10
Moribund	18	17	13	19
Natural deaths	2	3		3
Animals surviving to study termination	30	30	37	28
% probability of survival at end of study^b	60	60	74	56
Mean survival (days)	654	657	670	663
Survival analysis^c	P=0.946	P=1.000N	P=0.134N	P=0.924

^aCensored from survival analysis

^bKaplan-Meier determinations

^cThe result of the life table trend test (Tarone, 1975) is in the control column and the results of the life table pairwise comparisons (Cox, 1972) with the controls are in the exposed columns. A negative trend of lower mortality in an exposure groups is indicated by N.

 

Table 2: Mean body weights and survival

	0 ppm		3000 ppm			6000 ppm			12,000 ppm
Weeks on study	Av. wt (g)	No. of survivors	Av. wt (g)	Wt (% of controls)	No. of survivors	Av. wt (g)	Wt (% of controls)	No. of survivors	Av. wt (g)	Wt (% of controls)	No. of survivors
Males
Terminal sacrifice		27			34			30			33
Mean for weeks
1-13	245		240	98		237	97		228	93
14-52	399		389	97		388	97		373	93
53-101	442		429	97		432	98		417	97
Females
Terminal sacrifice		30			30			37			28
Mean for weeks
1-13	156		154	99		153	98		153	98
14-52	220		212	96		211	96		211	96
53-101	297		281	95		282	95		279	94

Conclusions:

Under the conditions of the study, there was no evidence of carcinogenic activity of the test substance in male or female rats receiving 3000, 6000 or 12,000 ppm; however rats may have tolerated higher doses.

Executive summary:

Groups of 60 male and 60 female F344/N rats were fed diets containing 0, 3000, 6000 or 12,000 ppm benzyl acetate for 2 years. Survival of exposed rats was similar to that of the controls. The mean bodyweights of the 12,000 ppm males and exposed females were approximately 5% lower than those of the controls throughout most of the study. The feed consumption by 12,000 ppm was slightly lower than the controls. Dietary levels of 3000, 6000 and 12,000 ppm benzyl acetate was estimated to result in average daily consumption levels of 130, 260 and 510 mg/kg bw (males) and 145, 290 and 575 mg/kg (females). No biologically significant changes in haematology or clinical chemistry parameters were found that could be attributed to benzyl acetate administration. No compound related increased incidences of neoplasms or nonneoplastic lesions occurred in males or females.

Reference 5

Endpoint:

carcinogenicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Information on the carcinogenic potential of the analogue read-across substance sodium benzoate was presented as a short summary, the information presented in this paper as part of the review of the available literature and the information is not sufficient to assess the quality of the result presented. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties. Sodium benzoate under physiological conditions is expected to dissociate to benzoic acid, a metabolite of benzyl acetate.

Principles of method if other than guideline:

Albino Swiss mice were dosed via drinking water with a high dose of sodium benzoate (2 %) from 5 weeks of age until death (the entire lifespan of the animal). Animals were assessed upon death for gross and histopathological changes.

GLP compliance:

not specified

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: In house colony- Age at study initiation: 5 weeks- Housing: Plastic cages in groups of 10 (separated by sex)- Diet: Regular pellets- Water: tap water ad libitum

Route of administration:

oral: drinking water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The solutions was prepared three times a week and administered in brown bottles to prevent degradation of the test chemical due to possible light sensitivity.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

From 5 weeks of age until the animals death.

Frequency of treatment:

Daily.

Remarks:

Doses / Concentrations:2 % in drinking waterBasis:nominal in water

Remarks:

Doses / Concentrations:119.2 mg/day for females and 124.0 mg/day for malesBasis:other: average daily intake (corresponds to 5.9 and 6.2 mL of drinking water consumed on average per day).

No. of animals per sex per dose:

50 animals per sex in the treatment group, 100 animals of each sex as untreated controls.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Prior to the carcinogenicity test, a toxicity study was performed with five dose levels of sodium benzoate. Eight mice per group were dosed with 8, 4, 2, 1 and 0.5 % sodium benzoate in drinking water daily for 35 days. Animals were assessed for survival, bodyweight, chemical consumption and histological changes. From this range finding study, the test concentration for the main test was selected as 2 %.

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes- Time schedule: WeeklyBODY WEIGHT: Yes- Time schedule for examinations: WeeklyWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes - Time schedule for examinations: The total consumption of water containing the test substance was measured when the fresh test substance solutions were prepared.

Sacrifice and pathology:

GROSS PATHOLOGY: YesAnimals were allowed to die or were sacrificed when moribund. Complete necropsies were performed on all animals and all organs were examined macroscopically.HISTOPATHOLOGY: YesHistological studies were performed on the liver, spleen, kidneys, bladder, thyroid, heart, pancreas, testes, ovaries, brain, nasal turbinates, at least four lobes of the lungs of each mouse and grossly altered organs.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITYSurvival rates of mice after weaning showed no treatment related effects.HISTOPATHOLOGY: No increase in the incidence of tumours was noted in the treated animals above that noted in the untreated control animals.ANALOGUE APPROACH JUSTIFICATION:- See attached “Justification for read-across” document for full details.- In summary, important considerations for the use of read-across for acute toxicity are: i) 1-phenylethyl acetate (the target chemical) has similar physico-chemical properties as benzyl acetate (the source substance) ii) there are structural similarities between the two chemicals, iii) the OECD QSAR Toolbox assigns an identical toxicity profiles to both chemicals, and iv) both chemicals have been tested for acute oral toxicity, which demonstrated that neither substance requires classification for acute toxicity and that the benzyl acetate will represent a worst-case scenario, and are adequate for classification and labelling and risk assessment purposes. Sodium benzoate under physiological conditions is expected to dissociate to benzoic acid, a metabolite of benzyl acetate.The information reported in this summary is included to demonstrate comparability between the source (sodium benzoate) and target (1-phenyl-ethyl acetate) substance.

Relevance of carcinogenic effects / potential:

Under the conditions of the test, no increase in the incidence of tumours was noted in the treated animals above the level recorded in the untreated controls. The study does not indicate any carcinogenic potential.

Dose descriptor:

NOEL

Effect level:

2 other: % solution

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Average total daily intake: Females 119.2 mgMales 124.0 mg

Remarks on result:

other: Effect type: toxicity

Table 1: Results

Treatment	Effective No. of Mice	Sex	Animals with tumours of:
			Lungs			Mal. Lymphomas			Blood Vessels			Other tissues^b
			No.	%	Age at death^a	No.	%	Age at death^a	No.	%	Age at death^a
2 % Sodium Benzoate	50	F	10	20	87 (51-134)	10	20	88 (57-119)	2	4	84 (55-113)	4 Adenocarcinomas of breast (44, 59, 78, 100); 1 carcinoma of skin (71); 1 adenoma of thyroid (134)
	50	M	7	14	85 (58-113)	3	6	81 (61-104)	4	8	81 (61-109)	1 Polyploid adenoma of cecum (61); 1 hepatoma (95)
Untreated Controls	99	F	21	21	95 (60-112)	24	24	89 (31-130)	5	5	112 (97-130)	2 Adenocarcinomas of breast (73, 93); 1 leiomyosarcoma of uterus (81); 2 adenocarcinomas of ovary (104, 106); 1 adenoma of glandular stomach (115); 1 adenoma of thyroid (116); 1 papilloma of oesophagus (106); 1 papilloma of skin (109), 1 firbosarcoma, subcutaneous (44); 1 adenoma of ovary (103); 1 malignant histiocytoma (98)
	99	M	23	23	95 (53-125)	12	12	90 (40-126)	6	6	88 (65-105)	1 Fibrosarcoma, subcutaneous (82); 1 papilloma of forestomach (63); 1 adenomacarcinoma of duodenum (113); 1 malignant histiocytoma (101); 1 carcinoma of skin (122); 1 adrenal cortical adenoma (130); 1 papilloma of oesophagus (63)
^aAverage and range in weeks ^bAge at death in weeks given in parentheses

 

Conclusions:

Sodium benzoate administered to mice in drinking water (as a 2 % solution) over the lifespan of male and female animals caused no detectable tumorgenic effect under the conditions of the test.

Executive summary:

Albino Swiss mice were dosed via drinking water with a high dose of sodium benzoate (2 %) from 5 weeks of age until death (the entire lifespan of the animal). Animals were assessed upon death for gross and histopathological changes. The average daily consumption of test substance was determined to be 119.2 mg/day for females and 124.0 mg/day for males (corresponds to 5.9 and 6.2 mL of drinking water consumed on average per day respectively). Sodium benzoate administered to mice in drinking water (as a 2 % solution) over the lifespan of male and female animals caused no detectable tumorgenic effect under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

510 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The key study is performed in line with sound scientific principles in line with a method comparable to an appropriate standardised guideline and is of sufficient quality with a Klimisch score of 2. Sufficient supporting studies are also available.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The combined conclusions of the two reports indicate that benzyl acetate has no carcinogenic effect. As benzyl acetate is being used to support the registration of 1-phenylethyl acetate on the basis of read-across it is considered that both substances can be considered as unclassified for carcinogenicity in line with Directive 67/548/EEC and Regulation 1272/2008.

Additional information

Two reports were available to assess the carcinogenic potential of benzyl acetate; both reports were published by NTP (1986 and 1993) and each contained a two year study on rats and mice with different methods of test substance application. The 1986 studies investigated the effect of benzyl acetate administered to mice and rats via oral gavage in corn oil. The method was similar to OECD 451 with some deviations thought not to affect the quality of the results; the in-life portion of the study was performed prior to GLP inception. The 1993 studies were also performed on mice and rats, however in this study the animals were dosed in feed in order to remove the potential influence the dosing methods exerted on the previous studies results. The latter study was performed to a method similar to OECD 451, with no discernible deviations from the standardised guideline; the study was performed in line with GLP. The second study also examined further the results and conclusions of the first study and indicated the underlying mechanism of the observed carcinogenicity in the previous study. Both studies were assigned a reliability score of 2 in accordance with Klimisch (1997). Due to the limited availability of data on 1-phenylethyl acetate, and the wealth of data available on benzyl acetate, the carcinogenicity of 1-phenylethyl acetate was assessed by reading-across between the two substances. The reported results with benzyl acetate are considered representative of the effects of 1-phenylethyl acetate. The substances are structurally very similar, the only difference being, benzyl acetate has no branching from the acetate. Both molecules have very similar physical-chemical properties and are anticipated to undergo the same metabolic pathway, and the simpler structure of benzyl acetate is likely to make it more toxicologically available and active and is therefore potentially a worse case of the effects of the registered substance.

In the 1986 NTP report, benzyl acetate was administered via gavage in corn oil in doses of 250 or 500 mg/kg bw to rats and 500 or 1000 mg/kg bw to mice. Administration of the test substance did not adversely affect survival or mean bodyweight gain. Survivals were generally comparable amongst groups; however only 15/50 (30%) of the control female mice and 18/50 (36%) of the low-dose female mice lived to the end of the study. An infection, resulting in suppurative inflammation or abscesses of the ovaries, uterus, mesentery, peritoneum or multiple organs was the probable cause of death in 26/35 control, 14/32 low-dose and 8/20 high-dose female mice before the end of the study. The incidence of ovarian abscesses and suppurative inflammation in the genital tract of female mice was inversely related to dose. Although the contribution of benzyl acetate to the decreased incidence of female mice with ovarian abscesses is not clear, this effect may be related to the bacteriostatic properties of its metabolites (benzyl alcohol and benzoic acid).

The increased incidence of high-dose male rats with retinopathy and cataracts or of low-dose females rats with retinopathy was not considered to be related to the test substance. These effects have been correlated at the test laboratory with the proximity of rats to fluorescent light. High-dose male and low-dose females were housed closest to the light source in this study.

Acinar-cell adenomas in the pancreas of male rats were considered to be related to the gavage administration of the test substance. A statistically significant positive trend was observed in the incidences of male rats with acinar-cell adenomas of the pancreas and the incidence in the high-dose group (37/49, 76%) was significantly higher than the incidence in vehicle controls (22/50, 44%). The incidence of acinar-cell tumours in the low-dose group (27/50) was not significantly greater than in the vehicle controls. The acinar-cell adenomas were more often multiple in the treated animals. Acinar-cell hyperplasia of the pancreas was observed in all dosed male rats (37/50 control; 34/50 low-dose; 36/49 high-dose) and, based on currently available information, this is considered to be due the the test substance despite concerns about the possibility of the influence from the vehicle or a combination of the vehicle and test substance.

Hepatocellular adenomas occurred with a statistically significant positive trend in male and female mice. Results of pairwise comparisons of the incidences for high-dose males and females were significant. The absence of hepatocellular adenomas in the concurrent female control groups may be due in part to early mortality; however, all adenomas in the high-dose group were observed between week 87 and the end of the study and 58% of the control females survived to at least this age. Since the incidences of high-dose males and females were significantly greater than those observed in concurrent controls and are also elevated relative to historical gavage controls for this laboratory (males: 36/298, 12.1%; females: 11/300, 3.7%), these increases are considered to be associated with administration of the test substance. The incidence of dosed male mice and high-dose female mice with hepatocellular carcinomas were elevated slightly but not significantly (male: control, 10/50, 20%; low-dose, 14/49, 29%; high-dose, 12/50, 24%; females: control, 1/50, 2%; low-dose, 0/50; high-dose, 4/50, 8%).

Squamous cell papillomas and squamous cell papillomas or carcinomas (combined) of the forestomach occurred with a positive trend in male mice; the incidence of squamous cell papillomas was also elevated in high-dose female mice. The increased incidence of forestomach tumours in both high-dose male and female mice was considerably higher than the historical corn oil gavage control rate at this laboratory (males: 2/296, 0.7%; females: 2/297, 0.7%) and throughout the NTP Carcinogenesis Program (males: 14/1070, 1.3%; females: 3/1073, 3%). Forestomach hyperplasia also occurred at increased incidences in dosed male and female mice. Both squamous cell papillomas or carcinomas and hyperplasia of the forestomach were probably associated with the administration of the test substance. Since the test substance was given by gavage and is a biologically reactive material, the increased incidence of hyperplasia in dosed mice may have been the result of direct action of the substance at the site of deposition in the forestomach.

Malignant preputial gland tumours occurred with a statistically significant positive trend in male rats. However, since the combined incidence of adenomas, adenocarcinomas or carcinomas was not statistically significant, these tumours are not considered to be associated with the administration of the test substance. The combined incidence of clitoral gland adenomas or carcinomas in female rats was not statistically increased. The incidences in high-dose rats of either sex (males: 6/50, 12%; females: 5/50, 10%) were higher than the corresponding historical corn oil gavage control rates at this laboratory (males: 11/300, 3.7%; females: 6/300, 2%) and throughout the Carcinogenesis Program (males: 42/1100, 3.8%; females: 27/1100, 2.5%).

Testicular (interstitial-cell) tumours in male rats were observed with a negative trend and the incidence in the high-dose group was lower than that in the controls. Conversely, there was an increase in the incidence of high-dose male rats with interstitial-cell hyperplasia. These two lesions are essentially similar differing mainly in size. Interstitial-cell tumours are generally larger with widespread necrosis. To better assess the potential relationship between the administration of the test substance and the incidence of male rats with these lesions, the combined incidence was considered more appropriate and was not statistically different from that of the controls. Neither the decrease in testicular tumours nor the increase in interstitial-cell hyperplasia was considered to be related to the administration of the test substance. Rather, the tumours appear to be associated with the aging process in these animals.

Cystic hyperplasia of the uterus/endometrium was observed in 26/50 (52%) control, 28/50 (56%) low-dose and 37/50 (74%) high-dose female mice. Because of the infection at this site, this increased incidence could not be clearly related to the administration of the test substance.

Under the conditions of this test, benzyl acetate increased the incidence of acinar-cell adenomas of the exocrine pancreas in male rats; the gavage vehicle may have been a contributing factor. No evidence of carcinogenicity was found for female rats. For male and female mice there was some evidence of carcinogenicity in that benzyl acetate caused increased incidences of hepatocellular adenomas and squamous cell neoplasms of the forestomach.

These conclusions caused much debate within the NTP expert panel that reviewed the findings. Much emphasis was placed on the potential influence of the corn oil vehicle and its known association with acinar-cell adenomas in rats and in mice, the deposition of the test substance in the stomach and the saturation of the metabolic pathway for benzyl acetate sparked considerable debate as potential mechanisms of the carcinogenic effect noted in this study.

In 1993, a second NTP report on the carcinogenicity of benzyl acetate was published. F344/N rats were fed diets containing benzyl acetate at 3000, 6000 and 12,000 ppm (calculated as 130, 260 and 510 mg/kg bw for males and 145, 290 and 570 mg/kg bw for females). Survival of exposed rats was similar to that of the controls. The mean bodyweights of the 12,000 ppm males and exposed females was approximately 5% lower than those of the control throughout most of the study. The feed consumption by 12,000 ppm males was slightly lower than controls. No biologically significant changes in chemistry were found that could be attributed to benzyl acetate administration. No compound related increased incidences of nonneoplastic lesions occurred in male or female rats.

A concurrent study was also performed on B6C3F1 mice. Male and female mice were fed diets containing 330, 1000 and 3000 ppm benzyl acetate (calculated as 35, 110 and 345 mg/kg bw for males and 40, 130 and 373 mg/kg bw for females). No biologically significant changes in haematology or clinical chemistry parameters were observed. No increase of neoplasms incidence in mice could be attributed to benzyl acetate administration. Dose-related increased incidences or severities of nonneoplastic nasal lesions occurred in the most posterior portions of the nasal cavity in all exposed groups. The lesions occurred in the majority of the exposed mice and consisted of atrophy and degeneration, primarily of the olfactory epithelium, cystic hyperplasia of the nasal submucosal glands, pigmentation of the mucosal epithelium and exudate accumulation. Under the conditions of this study, benzyl acetate did not exhibit carcinogenic effect in male and female mice and rats.

In the 1993 study, the conclusions of the 1986 report were revisited with new information from the 2 year feed studies and further data both on benzyl acetate and the vehicle used in the 1986 study, corn oil. The contrasting results in rats between the two NTP reports were discussed in much detail. From the available data it was apparent that high levels of fat administered to experimental animals enhanced the development of both spontaneous and chemically induced neoplasms. Various studies were available studying the effects of corn oil and also benzyl acetate in animals pre-treated with asazerine, a pancreatic carcinogen. The overall conclusion from the wealth of data available indicated that benzyl acetate is a weak promoter of carcinogen-induced pancreatic neoplasms and possibly spontaneous preneoplastic lesions.

The results from the experiments in mice had also produced contrasting results in the two NTP reports. A possible reason submitted for the difference in effects was the difference in dosing level. Animals dosed via gavage would be exposed to the total daily dose in one administration rather than a steady intake of the substance over 24 hours in a feed study. However a more likely mechanism was put forth, consistent with the findings of toxicokinetic data available on the administration of benzyl acetate. The study supporting the conclusions dosed rats and mice with benzyl acetate in corn oil by gavage at concentrations of 500 mg/kg in rats and 1000 mg/kg in mice for 7 days, also two further groups of mice and rats were dosed with 10,800 ppm and 2700 ppm respectively in diet for 7 days. The doses administered were considered comparable between the two methods, but the gavage administration saturated the benzoic acid elimination pathway, whereas dietary administration did not. Higher blood concentrations of benzyl acetate and its metabolites are likely via gavage. Both routes induced a depletion in the glycine supply and exerted the hippuric acid plasma concentrations were found to be comparable.

Concerning the nasal lesion in the 1993 study and the forestomach hyperplasia noted in the 1986 study, a comparison was drawn between the two effects. Benzyl acetate is a known respiratory tract irritant in humans, and causes intestinal irritation when ingested. The observed lesions are unsurprising when presented with this information. The forestomach is the site of deposition for gavage administration and is indicative of local effects. Similarly, the nasal lesions were considered to be due to the loss of the benzyl acetate from the food and a build up of the benzyl acetate vapour in the housing used in the study.

Overall, it can be concluded that benzyl acetate has no carcinogenic activity based on the data presented in the two NTP studies.

 

Supporting information was available in Toth (1984). The study was performed in line with good scientific principles, however the study was reported as a short communication, and lacked detail in the reporting throughout. The study was performed on the substance sodium benzoate, which is the sodium salt of benzoic acid (the sodium ion dissociates from the molecule within the body), the metabolite of benzyl acetate. Due to the lack of detail in the reporting, the study as assigned a reliability score of 4 in accordance with Klimisch (1997). In the study, Albino Swiss mice were dosed via drinking water with a high dose of sodium benzoate (2%) from 5 weeks of age until death (the entire lifespan of the animal). The average daily consumption of test substance was determined to be 119.2 mg/day for females and 124.0 mg/day for males (corresponds to 5.9 and 6.2 mL of drinking water consumed on average per day respectively). Sodium benzoate administered to mice in drinking water (as a 2% solution) over the lifespan of male and female animals caused no detectable tumorgenic effect under the conditions of the test.

Justification for selection of carcinogenicity via oral route endpoint:
Two studies available, one each on the rat and the mouse (see Discussion below).