Vinyl propionate

Carcinogenicity

Description of key information

No reliable data are available for Vinyl Propionate. However, reliable data from a closely related analogue Vinyl acetate (CAS 108-05-4) were used in a Read-across approach to fulfill this endpoint information requirement:

1. Literature data: Umeda, Y., et al., (2004). Carcinogenicity and chronic toxicity in mice and rats administered vinyl acetate monomer in drinking water. Journal of occupational health, 46(2), pp.87-99.

Result: positive for rats and mice. Good quality study, detailed information, according to OECD guideline 453.

2. Literature data: Bogdanffy, M.S., et al., (1994). Chronic toxicity and oncogenicity inhalation study with vinyl acetate in the rat and mouse. Toxicological Sciences, 23(2), pp.215-229.

Result: positive for rats and mice. Good quality study, detailed information, near guideline study(similar to OECD guideline 453).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

1981

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

Obtained form Wako Pure Chemical Industries (Osaka, Japan)
Five different lots.
Stored in dark vessels at room temperature

Species:

mouse

Strain:

other: Crj:BDF1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc., Shiga, Japan
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 4 Weeks
- Housing: individually in stainles steel wire-mesh hanging cages (112W x 212D x 120H mm)
- Diet: ad libitum CRF-1, Oriental Yeast Co. Ltd., Tokyo, Japan
- Water: ad libitum assigned drinking water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 15- 17
- Photoperiod (hrs dark / hrs light): 12h light/12 h dark

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

VEHICLE: water
- Concentration in vehicle: 0, 400, 2000 or 10'000 ppm

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas Chromatograph (Hewlett Packard HP5890A, USA) and Liquid chromatograph-mass spectrometer (Thermoquest TSQ-7000, USA)

Duration of treatment / exposure:

104 Weeks

Frequency of treatment:

Daily

Dose / conc.:

400 ppm (nominal)

Dose / conc.:

2 000 ppm (nominal)

Dose / conc.:

10 000 ppm (nominal)

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Daily observation for clinical signs and mortality.
Body weight and food and water consumption observed Weekly the first 13 weeks and every 4 weeks after that.
Urinary, hematological and blood biochemical parametes were determined on all surving animals on urine collected on week 104 and blood samples collected form the abdominal aortha under etherization after overnight fasting.
Urinary parameters analyzed usig Urolabstix (Diagnostic Diutsior, Bayer, Germany): pH, protein, glucose, ketone bodies, occult blood and urobilinogen.
Hematological and blood biochemical parametes were determined according to the OECD guideline

Sacrifice and pathology:

Organs were observed macroscopically and the tissues specified on the OECD guideline were subjected to hystopathological examinations.tissues were fixed in 10% neutral buffered formalin, and embedded in paraffin by the routine procedures. Tissue sections 5 microm thick were prepared and stained with hematoxylin and eosin. The oral cavity was examined histopathologically at six different levels.
The neoplastic and pre-neoplastic lesions were diagnosed with reference to the IARC monograph, the Standardized System of Nomenclature and Diagnostics Criteria and the Textbook of Oral Pathology.

Statistics:

Chi-square test was used for analysis of incidence of non-neoplastic lessions and urinary data.
Peto's Test and Fisher's exact test were used to analyze the incidence of neoplastic lessions.
Dunnett's test was used to analyze body weight, food and water consumption and hematological and blood biochemical parameters.
A lower confidence limit of the benchmark dose yielding a response with 10% extra risk (BMDL10) was calculated for combined incidences of squamous cell carcinomas and papillomas in the oral cavity.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Oral cavity nodules significantly increased in the10'000 ppm group of both sexes: three males (57 week) and five females (95 week)

Mortality:

mortality observed, treatment-related

Description (incidence):

6 males and 4 females (oral cavity tumor), 1 male (esophagus tumor), 1 male and 2 females (stomach tumor) and 1 male and 1 female (larynx tumor) on the 10'000 ppm group.
But no there was no significant difference in the survival rate between any test item treated group of each sex and the respective control animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight were significant lower by 30% in males and 18% in female animals that consumed 10'000ppm VA in water, respectively to the controls.

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption for both sex was lower in the 10000 ppm group, in particular in the last period of the 104 weeks.
Daily intake increased proportionally with the increase in the vinyl acetate concentration in drinking water, except for the 10'000 ppm group.
The estimated intake per body weight was higher in the female mice at all concentrations.

Daily vinyl acetate intake:
Males:
- control: 0 mg/kg
- 400: 42 mg/kg
- 2000: 202 mg/kg
- 10'000: 989 mg/kg
Females:
- control: 0 mg/kg
- 400: 63 mg/kg
- 2000: 301 mg/kg
- 10'000: 1418 mg/kg

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistical significant changes in absolute and relative organ weigth were observed on the 10'000 group (males and females). However there was no association with histopathological changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Mandibular nodules (in 3 males and 5 females) and maxillary nodules (3 males and 1 female) were observed in teh 10'000 group.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Incidence of squamous cell tumors in the oral cavity and forestomach increased significantly with increasing exposure in male and female animals.
Incidence of squamous cell tumors in the esophagous increased significantly with increasing exposure in male animals.
Squamous cell carcinomas increased significantly in those same organs in the 10'000 group.
Squamous cell carcinomas were observed in the larinx and esophagous of teh 10'000 group females, on the larinx of the 2000 group females and in the lariny of the 10'000 group males.
Sqamous cell papillomas were obseved in the oral cavity and stomach of 10'000 (male and female) group and in the esophagous of a 2000 group female.
The squamous cell papilloma was characterized by exophytic projection of the tumor tissue above the stratified squamous epithelium, which was composed of a central core of the turner connective tissue covered with hyperplastic squamous cells having neither cellular nor structural atypia.
Squamous cell tumors having either cellular or structural atypia were diagnosed as squamous cell carcinoma. Many of those were characterized by endophytic growth in which the tumor cells were proliferated inward into the subepithelial tissue.
The endophytic growth type of squamous cell carcinomas, had invasion of the tumor tissue into the muscle layer that was indicative of severe malignancy. Moreover, 5 male mice were found to bear squamons cell carcinomas in multiple organs.
8 squamous cell carcinomas were metastasized into the lungs, the pancreas, the liver,the kidneys, and the lymph nodes.

Pre-neoplastic lesions were observed in the stralified squamous epilhelium of the upper digeslive tract, and were further classified into basal cell hyperplasia, squamous cell hyperplasia and epithelial dysplasia.
In the oral cavity, basal cell hyperplasia occured in the 2'000 and 10'000 ppm group in both sexes, and the epithelial dysplasia increased markedly in the 10'000 ppm group of both sexes.

Other types of tumors were observed in the spleen, lungs, uterus and nasal cavity of the vinyl acetate-administered groups, but the incidences of those tumors were not statistically different from those in the controls

Key result

Dose descriptor:

BMDL10

Effect level:

477 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Dose-response relationship belween estimated daily vinyl acetate intake (mg/kg/d) and incidence of oral cavity squamous cell lumors

Conclusions:

This study presents clear evidence of a dose-relaled increase in the incidences of malign and benign squamous cell tumors in the upper digeslive tract of the mice of both sexes when Vinyl acetate is orally administered in drinking water for two years.
A lower confidence limit of a benchmark dose (BMDL10) of 477 mg/k/d was calculated based on a dose-response relationship between combined incidence of squamous cell carcinomas and papillomas in the oral cavity and the estimated daily Vinyl acetate intakes per body weight.

Executive summary:

Carcinogenicity and chronic toxicity ot vinyl acetate were examined in male and female Crj:BDF mice. 50 mice per group of each sex were orally administered Vinyl acetate in drinking water containing 0, 400, 2'000 or 10'000 ppm during 104 weeks.

Squamous cell tumors found in the upper digestive tract and in the larynx of treated mice of both sexes. Squamous cell carcinomas and papillomas were observed in the oral cavity, esophagus, forestomach and larynx ot the 10,000 ppm group, together with basal cell hyperplasia, squamous cell hyperplasia and epithelial dysplasia. Pre-neoplastic hyperplasias were also seen.

A lower confidence limit of a benchmark dose (BMDL₁₀) of 477 mg/k/d was calculated based on a dose-response relationship between combined incidence of squamous cell carcinomas and papillomas in the oral cavity and the estimated daily Vinyl acetate intakes per body weight.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The substances discussed in this analogue approach are vinyl propionate (target substance) and vinyl acetate (source substance), where both are hypothesized to be readily metabolized [(bio)transformation to a common compound – Scenario 1 of the RAAF guidance] to acetaldehyde (common compound and putative agent) and their corresponding carboxylic acids.
The hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate , leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Vinyl acetate is a high purity substance (99.8% w/w) with a negligible amount of impurities [water (= 0.03 – 0.1%, w/w), acetic acid (= 0.005 – 0.01%, w/w), acetaldehyde (= 0.005 – 0.02%, w/w)] (ECHA 2008). Similarly, vinyl propionate is a high purity substance (99.88%) (Gamer et al. 1997; Intertek 2017), with a negligible amount of impurities [water (= 0.05%)]. The scientific aspects of this Assessment Element have been addressed and the supporting evidence is “acceptable with high confidence”.

3. ANALOGUE APPROACH JUSTIFICATION
The read-across hypothesis and justification establish the structural similarities and differences of the source and target substances. The similarities in their structures and metabolic pathways serve as the foundation for this read-across and justify prediction of vinyl propionate’s toxicological properties from vinyl acetate. The structural differences between the source and target substance are not expected to be linked to differences in toxicological properties. The information reported in Sipi et al. (1992), Chahinian et al. (2002, 2010), the OECD QSAR toolbox’s metabolism/transformation profiler (v4.2, 2018), and well-established theoretical understanding of ester hydrolysis (Adams et al. 2008) provides ample evidence to support acceptability of this Assessment Element. The information provided by these studies also provide strong evidence that the same types of effects are caused in the same biological targets by the common compounds (acetaldehyde and acetic acid). The scientific aspects of this AE have been addressed and the supporting evidence is “acceptable with high confidence”.

The detailed justification for the analogue approach is added to section 13 of this dossier.

4. DATA MATRIX
The detailed data Matrix for the analogue approach is added to section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

BMDL10

Effect level:

555 mg/kg bw/day

Based on:

other: Read across from analogue Vinyl Acetate

Sex:

male/female

Basis for effect level:

other: Dose-response relationship belween estimated daily vinyl acetate intake (mg/kg/d) and incidence of oral cavity squamous cell lumors

Remarks on result:

other: Calculated from BMDL10 for Vinyl Acetate : 447 mg/kg bw day(MW 86.1), taking into account the MW for Vinyl propionate (MW: 100.1)

Conclusions:

There is clear evidence of a dose-relaled increase in the incidences of malign and benign squamous cell tumors in the upper digeslive tract of the mice of both sexes when Vinyl acetate is orally administered in drinking water for two years.
A lower confidence limit of a benchmark dose (BMDL10) of 447 mg/k/d was calculated based on a dose-response relationship between combined incidence of squamous cell carcinomas and papillomas in the oral cavity and the estimated daily Vinyl acetate intakes per body weight.

Based on this read-across, vinyl propionate is classified as carcinogen 2 (H351) according to Regulation (EC) No 1272/2008 (CLP regulation) and no further testing for this endpoint is required.
The BMDL10 for Vinyl propionate was calculated to be 555 mg/kg bw d.

Executive summary:

Vinyl propionate was assessed based on read-across from vinyl acetate.

As explained in the justification for type of information, the hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate, leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group. The similarities in their structures and metabolic pathways serve as the foundation for this read-across and justify prediction of vinyl propionate’s toxicological properties from vinyl acetate.

A lower confidence limit of a benchmark dose (BMDL10) of 477 mg/k/d was calculated based on a dose response relationship between combined incidence of squamous cell carcinomas and papillomas in the oral cavity and the estimated daily Vinyl acetate (source) intakes per body weight.

Based on this read-across, vinyl propionate is classified as carcinogen 2 (H351) according to Regulation (EC) No 1272/2008 (CLP regulation) and no further testing for these endpoint is required.

The BMDL10 for Vinyl propionate was calculated to be 555 mg/kg bw d.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

1981

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

Obtained form Wako Pure Chemical Industries (Osaka, Japan)
Five different lots.
Stored in dark vessels at room temperature

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc., Kanagawa, Japan
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 4 Weeks
- Fasting period before study: none
- Housing: individually in stainles steel wire-mesh hanging cages (170W x 294D x 176H mm)
- Diet: ad libitum CRF-1, Oriental Yeast Co. Ltd., Tokyo, Japan
- Water: ad libitum assigned drinking water
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 15- 17
- Photoperiod (hrs dark / hrs light): 12h light/12 h dark

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

VEHICLE: water
- Concentration in vehicle: 0, 400, 2000 or 1000 ppm

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas Chromatograph (Hewlett Packard HP5890A, USA) and Liquid chromatograph-mass spectrometer (Thermoquest TSQ-7000, USA)

Duration of treatment / exposure:

104 Weeks

Frequency of treatment:

Daily

Dose / conc.:

400 ppm (nominal)

Dose / conc.:

2 000 ppm (nominal)

Dose / conc.:

10 000 ppm (nominal)

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Daily observation for clinical signs and mortality.
Body weight and food and water consumption observed Weekly the first 13 weeks and every 4 weeks after that.
Urinary, hematological and blood biochemical parametes were determined on all surving animals on urine collected on week 104 and blood smaples collected form the abdominal aortha under etherization after overnight fasting
Urinary parameters analyzed usig Urolabstix (Diagnostic Diutsior, Bayer, Germany): pH, protein, glucose, ketone bodies, occult blood and urobilinogen.
Hematological and blood biochemical parametes were determined according to the OECD guideline

Sacrifice and pathology:

Organs were observed macroscopically and the tissues specified on the OECD guideline were subjected to hystopathological examinations.tissues were fixed in 10% neutral buffered formalin, and embedded in paraffin by the routine procedures. Tissue sections 5 microm thick were prepared and stained with hematoxylin and eosin. The oral cavity was examined histopathologically at six different levels.
The neoplastic and pre-neoplastic lesions were diagnosed with reference to the IARC monograph, the Standardized System of Nomenclature and Diagnostics Criteria and the Textbook of Oral Pathology.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In a 400 ppm female an oral cavity nodule appeared after 87 weeks.

Mortality:

mortality observed, treatment-related

Description (incidence):

2 male rats (oral cavity tumors) in the 10'000 ppm group. But there was no statistical significant difference on the survival rate between any test item trated animal and the respective control animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of the 10'000 ppm were significantly lower by 8% in males and by 10% in females respectively to the control animals at the end of the experiment.

Food consumption and compound intake (if feeding study):

no effects observed

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption of the 10'000 ppm were significantly lower by 15% in males and by 18% in females respectively to the control animals thourgout the experimental period.
The estimated daily vinyl acetate intake per body weight increased with an increase in the vinyl acetate concentration in the drinking water except for the 10,000 ppm group.

Daily vinyl acetate intake:
Males:
- control: 0 mg/kg
- 400: 21 mg/kg
- 2000: 98 mg/kg
- 10'000: 442 mg/kg
Females:
- control: 0 mg/kg
- 400: 31 mg/kg
- 2000: 146 mg/kg
- 10'000: 575 mg/kg

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistical significant changes in absolute and relative organ weigth were observed on the 10'000 group (males and females). However there was no association with histopathological changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Mandiblar nodules (3 males in the 10'000 ppm group, and a female in the 400 ppm group). Maxillary nodules (one female in the 10'000 ppm group).

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Squamous cell carcinomas and squamous cell papillomas were observed in the oral cavity and the esophagus.
The squamous cell papilloma was characterized by exophytic projection of the tumor tissue above the stratified squamous epithelium, which was composed of a central core of the turner connective tissue covered with hyperplastic squamous cells having neither cellular nor structural atypia.
Squamous cell tumors having either cellular or structural atypia were diagnosed as squamous cell carcinoma. Many of those were characterized by endophytic growth in which the tumor cells were proliferated inward into the subepithelial tissue.
Squamous cell carcinomas and papillomas were seen in the oral cavity of 5 and 2 10,000 ppm male animals, reapectively. The incidence was significantly higher than that in the control group, and increased in a treatment-related manner.
Squamous cell carcinomas were observed in the oral cavity of 3 female rats and in the esophagus of 1 female rat in Ihe 10'000 ppm group.The incidence of oral cavity and esophagus squamous cell carcinomas exceeded the range of the historical control.
One oral cavity squamous cell carcinoma was also seen in the 2'000 ppm group and one in the 400 ppm female group.
Most squamous cell carcinomas except two had endophytic growth which was characterized by proliferative invasion of the tumor tissue inward into the subepithelial tissue. Of those endophytic carcinomas, 3 had invasion of the tumor tissue into the muscle layer, indicative of severe malignancy.
Pre-neoplastic lesions were observed in the stratified squamous epithelium of the upper digestive tract. All the pre-neoplastic lesions occurred in the 10'000
ppm group except for the forestomach squamous cell hyperplasia in a control and a 2'000 ppm male

Key result

Dose descriptor:

BMDL10

Effect level:

447 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Dose-response relationship belween estimated daily vinyl acetate intake (mg/kg/d) and incidence of oral cavity squamous cell lumors

Conclusions:

This study presents clear evidence of a dose-relaled increase in the incidences of malign and benign squamous cell tumors in the upper digeslive tract of the rat of both sexes when Vinyl acetate is orally administered in drinking waler for two years.
A lower confidence limit of a benchmark dose (BMDL10) of 477 mg/k/d was calculated based on a dose-response relationship between combined incidence of squamous cell carcinomas and papillomas in the oral cavity and the estimated daily Vinyl acetate intakes per body weight.

Executive summary:

Carcinogenicity and chronic toxicity ot vinyl acetate were examined in male and female F344/DuCrj rats. 50 rats per group of each sex were orally administered Vinyl acetate in drinking water containing 0, 400, 2'000 or 10'000 ppm during 104 weeks.

Squamous cell tumors were observed in the upper digestive tract treated rats, in both sexes. Incidences of squamous cell carcinomas and papillomas were increased in the oral cavity of the 10'000 ppm group of both sexes, and an esophagus

squamous cell carcinoma was observed in a 10'000 ppm female. Pre-neoplastic hyperplasias were also found in both sexes in the oral cavity. A lower confidence limit of a benchmark dose (BMDL₁₀) of 477 mg/k/d was calculated based on a dose-response relationship between combined incidence of squamous cell carcinomas and papillomas in the oral cavity and the estimated daily Vinyl acetate intakes per body weight.

Reference 4

Endpoint:

carcinogenicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The substances discussed in this analogue approach are vinyl propionate (target substance) and vinyl acetate (source substance), where both are hypothesized to be readily metabolized [(bio)transformation to a common compound – Scenario 1 of the RAAF guidance] to acetaldehyde (common compound and putative agent) and their corresponding carboxylic acids.
The hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate , leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Vinyl acetate is a high purity substance (99.8% w/w) with a negligible amount of impurities [water (= 0.03 – 0.1%, w/w), acetic acid (= 0.005 – 0.01%, w/w), acetaldehyde (= 0.005 – 0.02%, w/w)] (ECHA 2008). Similarly, vinyl propionate is a high purity substance (99.88%) (Gamer et al. 1997; Intertek 2017), with a negligible amount of impurities [water (= 0.05%)]. The scientific aspects of this Assessment Element have been addressed and the supporting evidence is “acceptable with high confidence”.

3. ANALOGUE APPROACH JUSTIFICATION
The read-across hypothesis and justification establish the structural similarities and differences of the source and target substances. The similarities in their structures and metabolic pathways serve as the foundation for this read-across and justify prediction of vinyl propionate’s toxicological properties from vinyl acetate. The structural differences between the source and target substance are not expected to be linked to differences in toxicological properties. The information reported in Sipi et al. (1992), Chahinian et al. (2002, 2010), the OECD QSAR toolbox’s metabolism/transformation profiler (v4.2, 2018), and well-established theoretical understanding of ester hydrolysis (Adams et al. 2008) provides ample evidence to support acceptability of this Assessment Element. The information provided by these studies also provide strong evidence that the same types of effects are caused in the same biological targets by the common compounds (acetaldehyde and acetic acid). The scientific aspects of this AE have been addressed and the supporting evidence is “acceptable with high confidence”.

The detailed justification for the analogue approach is added to section 13 of this dossier.

4. DATA MATRIX
The detailed data Matrix for the analogue approach is added to section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

BMCL10

Effect level:

555 mg/kg bw/day

Based on:

other: Read across from analogue Vinyl Acetate

Sex:

male/female

Basis for effect level:

other: Dose-response relationship belween estimated daily vinyl acetate intake (mg/kg/d) and incidence of oral cavity squamous cell lumors

Remarks on result:

other: Calculated from BMDL10 for Vinyl Acetate : 447 mg/kg bw day(MW 86.1), taking into account the MW for Vinyl propionate (MW: 100.1)

Conclusions:

There is clear evidence of a dose-relaled increase in the incidences of malign and benign squamous cell tumors in the upper digeslive tract of the rat of both sexes when Vinyl acetate is orally administered in drinking waler for two years.
A lower confidence limit of a benchmark dose (BMDL10) of 477 mg/k/d was calculated based on a dose response relationship between combined incidence of squamous cell carcinomas and papillomas in the oral cavity and the estimated daily Vinyl acetate (source) intakes per body weight.

Based on this read-across, vinyl propionate is classified as carcinogen 2 (H 351) according to Regulation (EC) No 1272/2008 (CLP regulation) and no further testing for this endpoint is required.
The BMDL10 for Vinyl propionate was calculated to be 555 mg/kg bw d.

Executive summary:

Vinyl propionate was assessed based on read-across from vinyl acetate.

As explained in the justification for type of information, the hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate, leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group. The similarities in their structures and metabolic pathways serve as the

foundation for this read-across and justify prediction of vinyl propionate’s toxicological properties from vinyl acetate.

A lower confidence limit of a benchmark dose (BMDL10) of 477 mg/k/d was calculated based on a dose response relationship between combined incidence of squamous cell carcinomas and papillomas in the oral cavity and the estimated daily Vinyl acetate (source) intakes per body weight.

Based on this read-across, vinyl propionate is classified as carcinogen 2 (H351) according to Regulation (EC) No 1272/2008 (CLP regulation) and no further testing for these endpoint is required.

The BMDL10 for Vinyl propionate was calculated to be 555 mg/kg bw d.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

BMDL10

555 mg/kg bw/day

Study duration:

chronic

Species:

other: mouse and rat

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

not applicable

GLP compliance:

yes

Specific details on test material used for the study:

Eleven batches received from BP Chemicals. Ltd, (UK)

Species:

mouse

Strain:

other: CrI:CD-I(ICR)BR

Remarks:

Swiss mouse-derived outbred strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd., (Margate. UK)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 28 days
- Weight at study initiation: males: 22.4 to 35.5 g, females: 16.0 to 28.5 g.
- Fasting period before study: none
- Housing: in a single room, caged individually on stainless steel cages suspended over cardboard-lined trays.
- Diet: ad libitum SQC Rat and Mouse Maintenance Diet No. 1, Expanded (Special Diets Services. LTD,. Witham. UK) except during exposure and overnight prior to blood sample collection
- Water: ad libitum except during exposure
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): min. 15
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 8m2 chambers constructed of laminated glass and milled steel.
- Method of conditioning air: filtration
- System of generating particulates/aerosols: liquid vinyl acetate was nebulized within an expansion chamber of a generator module. The aerosol produced was mixed with filtered exposure chamber air and introduced into the chamber as a vapor.
- Air flow rate: 1500 to 2000 l/min
- Chamber temperature: between 19 and 25°C.
- Chamber humidity: between 40 and 80%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Vinyl acetate was measured in all chambers every 15 min with a Sigma 4 gas chromatograph (Perkin-Elmer. Ltd. Beaconsfield, UK) fitted with a single-loop gas injection valve and flame ionization detector. The gas chromatograph conditions were: 0.5-m Carbowax 1540. 1/4.in. i.d. stainless steel column, nitrogen carrier gas, column temp. 80°C and injector temp.300°C.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

6 h, 5 days per week

Post exposure period:

Only for Recovery satelite group: 70 weeks exposure followed by 15 (males) or 16 (females) weeks of recovery

Dose / conc.:

50 ppm (nominal)

Remarks:

Mean measured concentration: 49.4 +/- 2.4 ppm

Dose / conc.:

200 ppm (nominal)

Remarks:

Mean measured concentration: 200.5 +/- 9.7 ppm

Dose / conc.:

600 ppm (nominal)

Remarks:

Mean measured concentration: 594.7 +/- 16.8 ppm

No. of animals per sex per dose:

60 for oncogenic studies
10 Satelite group for clinical laboratory evaluation (week 51) and ad-interim euthanasia (week 52-53)
10 Satelite group for clinical laboratory evaluation (week 81) and ad-interim euthanasia (week 85-86)
10 Satelite group for recovery (70 week exposure followed by 15 weeks (males) and 16 weeks (females) exposure free)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 3-months study with exposure concentrations of 50, 200 and 1000 ppm.
- Animal assignment: random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- twice daily for morbidity/mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: brief observation before each exposure, detailled observations once weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly intervals until week 28 and every 4 weeks thereafter. For recovery animals weekly from week 71 to 74 and every 4 weeks thereafter.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 51 and 81 for satelite groups 1 and 2 and week 104 for main group
- Blood collected by orbital sinus puncture
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Yes, overnight
- How many animals: 10 (satelite groups) and 10 animals from main groups
- Parameters checked:
Haematology: hemoglobin, mean cell volume, red blood cell! count, mean cell hemoglobin, packed cell volume, mean cell hemoglobin concentration, total and differential white blood cell count, and prothrombin time.
Clinical chemistry: glutamate oxalale transaminanase, glutamate pyruvate transaminase, alkaline phosphatase, creatinine phosphokinase, sodium, chloride, potasium, calcium, glucose, blood urea nitrogen, albumin, total protein and albumin/globin ratio.

Sacrifice and pathology:

Animals were euthanized by an intraperitoneal injection of sodium pentobarbitone and exanguination following an overnight fast.

GROSS PATHOLOGY: Yes
organ weight: adrenals, gonads, kidneys, lungs, spleen, brain, heart, liver and thyroids.

HISTOPATHOLOGY: Yes
Eyes which were fixed in Davidson's fluid, the bone marrow smear was fixed in methanol, testes were fixed in Bouin's solution, all other tissue samples were fixed in 10% neutral buffered formalin.
Adipose tissue, adrenals, aorta, bone marrow smear, brain (medullary, cerebellar, and cortical section), cecum, duodenum, ear canal, esophagus, eyes (and optic nerve), fallopian tubes, femur, ganglia (Iumbar, sacral and dorsal), Harderian glands, heart, hind limbs, (postdistal portions for examination of tibial and plantar nerves), ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (mandibular, mediastinal, mesenteric, und bronchial), mammary gland, muscle (quadriceps), nasal turbinates (six levels), ovaries, pancreas, pituitary, prostate, rectum, salivary gland (submaxillary), sciatic nerves, seminal vesicles, skin, spinal cord (high cervical), spleen, stomach (glandular and forestomach), testes with epididymides, thymus (where present), thyroids (with parathyroids where identified), tonge, trachea, urinary bladder, uterus (including cervix), and gross lesions and tissue masses.

Two histopathological evaluations were performed. The first at Hazleton Laboratories, UK. A second, independent. and more detailed review of the respiratory tract tissues was performed at the TNO-ClVO laboratories in the Netherlands.

Statistics:

Body weights and body weight gains, heamatology parameters, measured clinical chemistry parameters, urine volume, terminal body and organ weights, and relative organ weights: One-way analysis of variance followed by pairwise t tests of treated groups against controls.
Urine specific gravity and pH: Kruskal-Wallis test followed by the Wilcoxon rank·sum test.
Survival probabilily functions: Kaplan-Meier technique and compared by the Iog-rank procedure.
Tumor incidence data: evaluated across groups using the Cochran-Armitage trend test.
Nonneoplastic and neoplastic lesions: evaluated between groups using the Fisher's exact test.
Unless otherwise stated, statistical significance was judged at the p=<0.05 level.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Rough haircoat and hunched posture were observed at all concentrations.

Description (incidence):

No consistent increase in mortality associated with treatment was observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reduction in weight gain in 600 ppm groups: 15% below controls. In 200 ppm groups there was also a significant decrease of weight gain.
Male mice in the 600 ppm group and female mice in all exposure groups showed statistically significant increases in weight gain during the recovery period.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increases in absolute and relative lung weights of mice at Week 53 were observed on the 600 ppm males.
At week 83, these changes were seen in the 200 and 600 ppm males and 600 ppm females.
At week 104 significant increases were observed in 600 ppm males and females.
In the recovery groups there were no statistically significant differences between the groups.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Atrophic areas of the olfactory epithelium were accompanied by foci of respiratory epithelium (respiratory metaplasia).
In the 600 ppm group, focal non keratinizing squamous metaplasia of respiratory epithelium of the maxilloturbinates and lateral wall of the nasal cavity at the naso/maxilloturbinate region, and occasionally of olfactory epithelium at the dorsal meatus, was observed. Also, eosinophilic hypenrophic sustentacular cells along with local loss of sensory cells was observed in all groups, including controls, but occurred more frequently in mice of the 200 and 600 ppm groups.
Tracheal epithelial hyperplasia was significantly increased in incidence in 600 ppm groups.

In the 600 group flaltening and/or exfoliation of the bronchial and bronchiolar lining epithelium, without obvious evidence of an associated inflammatory response was observed. Intraluminal fibroepitheIial projections, seen as finger-like projections, plaques and buds, protruding into the lumen of the bronchi and bronchioli were also observed. The projections were lined by flattened epithelium and they incorporated a stromal component.

Epithelial disorganization of the bronchial and bronchiolar epithelium was defined as the presence foci or areas of dedifferentiated lining epithelium, seen as a pleomorphic picture of swollen basopilic epithelial cells showing pronounced nucleoli, together with relatively flaltened or cuboidal epithelial foci, suggesting regeneration, and occasionally multilayered or hyperplastic foci. Focal metaplasia of the bronchi/bronchioli was occasionally observed.
In 1 male of the 600 ppm group a small area in the alveolar tissue with cornifying squamous metaplasia was observed.

In 600 ppm females there were compound-related accumulations of alveolar (foamy) macrophages.
In the 600 ppm group in both sexes and also in males of the 200 ppm group accumulation of brown-pigmented macrophages was observed, as well as intraalveolar accumulation of eosinophilic material.

1 female in the 600 ppm group showed a focus of squamous epithelial hyperplasia with dysplastic changes. Several other females showed epithelial hyperplasia.

Nonneoplastic changes were similar in the recovery groups.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A single moderately invasive squamous cell carcinoma was found in a major bronchus of the lung of a male of the 600 ppm group while a single adenocarcinoma occurred in a male of the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

176 mg/m³ air

Based on:

other: Calculation

Remarks:

Conversion factor: 1ppmv = 3.52 mg/m3 Based on MW of 86.09, 25 °C, 1 atmosphere

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Conclusions:

Inhalation exposure to vapours of vinyl acetate for 104 weeks, lead to non neoplastic lesions of the nasal cavity on mice at 200 and 600 ppm. The non observable adverse effect level was determined to be 50 ppm (176 mg/m3).

Executive summary:

Chronic toxicity and oncogenicity of vinyl acelate in male and female mice was evaluated in a 104-week study.

Exposure concentrations were 0, 50, 200, and 600 ppm. The study included satelite groups for terminations at approximately 53 and 83 weeks and a recovery group (exposure for 70 weeks and 15-week recovery).

Body weight gain was statistically significant reduced in the 200 and 600 ppm groups. Recovery animals showed significant improvements in weight gain relative to controls.

No changes in hematological or clinical chemical parameters were observed that could be unequivocally related to treatment.

There were no adverse effects on survival.

Increases in lung weight were observed in the 600 ppm groups. These changes were associated with bronchial exfoliation, macrophage accumulation, and fibrous plaques and buds extending into the airway lumen, and bronchial/bronchialar epithelial disorganization.

The most significant histopathological changes were noted in the nasal cavity at concentrations of 200 and 600 ppm. In the olfactory epithelium, the main non-neoplastic changes included epithelial atrophy, regenerative effects (squamous metaplasia and respiratory metaplasia of olfactory epithelium), basal cell hyperplasia, and epithelial nest-like infolds. Squamous metaplasia at the nasal maxilloturbinate region was prevalent in mice.

Nonneoplastic changes were similar in the recovery groups.

One squamous cell carcinoma was found in the lung of a mouse of the 600 ppm group.

The no-observable adverse-effect level for all effects (systemic and histopathological) was determined to be 50 ppm (176 mg/m³).

The tumorigenic response appears to be nonlinear.

Reference 2

Endpoint:

carcinogenicity: inhalation

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The substances discussed in this analogue approach are vinyl propionate (target substance) and vinyl acetate (source substance), where both are hypothesized to be readily metabolized [(bio)transformation to a common compound – Scenario 1 of the RAAF guidance] to acetaldehyde (common compound and putative agent) and their corresponding carboxylic acids.
The hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate , leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Vinyl acetate is a high purity substance (99.8% w/w) with a negligible amount of impurities [water (= 0.03 – 0.1%, w/w), acetic acid (= 0.005 – 0.01%, w/w), acetaldehyde (= 0.005 – 0.02%, w/w)] (ECHA 2008). Similarly, vinyl propionate is a high purity substance (99.88%) (Gamer et al. 1997; Intertek 2017), with a negligible amount of impurities [water (= 0.05%)]. The scientific aspects of this Assessment Element have been addressed and the supporting evidence is “acceptable with high confidence”.

3. ANALOGUE APPROACH JUSTIFICATION
The read-across hypothesis and justification establish the structural similarities and differences of the source and target substances. The similarities in their structures and metabolic pathways serve as the foundation for this read-across and justify prediction of vinyl propionate’s toxicological properties from vinyl acetate. The structural differences between the source and target substance are not expected to be linked to differences in toxicological properties. The information reported in Sipi et al. (1992), Chahinian et al. (2002, 2010), the OECD QSAR toolbox’s metabolism/transformation profiler (v4.2, 2018), and well-established theoretical understanding of ester hydrolysis (Adams et al. 2008) provides ample evidence to support acceptability of this Assessment Element. The information provided by these studies also provide strong evidence that the same types of effects are caused in the same biological targets by the common compounds (acetaldehyde and acetic acid). The scientific aspects of this AE have been addressed and the supporting evidence is “acceptable with high confidence”.

The detailed justification for the analogue approach is added to section 13 of this dossier.

4. DATA MATRIX
The detailed data Matrix for the analogue approach is added to section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

other: Read across from analogue Vinyl Acetate

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

205 mg/m³ air

Based on:

other: Read-across from analogue Vinyl Acetate

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Remarks on result:

other: Calculated from NOAEL for Vinyl Acetate : 50ppm, Conversion factor: 1ppmv = 4.1 mg/m3 Based on MW of 100.12 (for Vinyl propionate), 25 °C, 1 atmosphere

Conclusions:

Inhalation exposure to vapours of vinyl acetate for 104 weeks, lead to non neoplastic lesions of the nasal cavity on mice at 200 and 600 ppm. The non observable adverse effect level was determined to be 50 ppm (176 mg/m3).

The non observable adverse effect level for Vinyl Propionate was calculated to be 205 mg/m3 (50 ppm).

Based on this read-across, vinyl propionate is classified as carcinogen 2 (H351) according to Regulation (EC) No 1272/2008 (CLP regulation) and no further testing for this endpoint is required.

Executive summary:

Vinyl propionate was assessed based on read-across from vinyl acetate.

As explained in the justification for type of information, the hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate, leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group. The similarities in their structures and metabolic pathways serve as the

foundation for this read-across and justify prediction of vinyl propionate’s toxicological properties from vinyl acetate.

Inhalation exposure to vapours of vinyl acetate for 104 weeks, lead to non neoplastic lesions of the nasal cavity on mice at 200 and 600 ppm. The non observable adverse effect level was determined to be 50 ppm (176 mg/m³).

The non observable adverse effect level was calculated for Vinyl propionate to be 205 mg/m3 (50 ppm).

Based on this read-across, vinyl propionate is classified as carcinogen 2 (H351) according to Regulation (EC) No 1272/2008 (CLP regulation) and no further testing for these endpoint is required.

Reference 3

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

not applicable

GLP compliance:

yes

Specific details on test material used for the study:

Eleven batches received from BP Chemicals. Ltd, (UK)

Species:

rat

Strain:

other: Crl:CD(SD)BR

Remarks:

Sprague-Dawley-derived outbred strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd., (Margate. UK)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 28 days
- Weight at study initiation: males: 115.5 to 243.2 g, females: 127.6 to 189.1 g.
- Fasting period before study: none
- Housing: in a single room, caged in groups of five by sex, on stainless steel cages suspended over cardboard-lined trays.
- Diet: ad libitum SQC Rat and Mouse Maintenance Diet No. 1, Expanded (Special Diets Services. LTD,. Witham. UK), except during exposure and overnight prior to blood sample collection
- Water: ad libitum, except during exposure
- Acclimation period: 17 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): min. 15
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 8m2 chambers constructed of laminated glass and milled steel.
- Method of conditioning air: filtration
- System of generating particulates/aerosols: liquid vinyl acetate was nebulized within an expansion chamber of a generator module. The aerosol produced was mixed with filtered exposure chamber air and introduced into the chamber as a vapor.
- Air flow rate: 1500 to 2000 l/min
- Chamber temperature: between 19 and 25°C.
- Chamber humidity: between 40 and 80%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Vinyl acetate was measured in all chambers every 15 min with a Sigma 4 gas chromatograph (Perkin-Elmer. Ltd. Beaconsfield, UK) fitted with a single-loop gas injection valve and flame ionization detector. The gas chromatograph conditions were: 0.5-m Carbowax 1540. 1/4.in. i.d. stainless steel column, nitrogen carrier gas, column temp. 80°C and injector temp.300°C.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

6 h, 5 days per week

Dose / conc.:

50 ppm (nominal)

Remarks:

Mean measured concentration: 49.4 +/- 2.4 ppm

Dose / conc.:

200 ppm (nominal)

Remarks:

Mean measured concentration: 200.5 +/- 9.7 ppm

Dose / conc.:

600 ppm (nominal)

Remarks:

Mean measured concentration: 594.7 +/- 16.8 ppm

No. of animals per sex per dose:

60 for oncogenic studies
10 Satelite group for clinical laboratory evaluation (week 51) and ad-interim euthanasia (week 52-53)
10 Satelite group for clinical laboratory evaluation (week 81) and ad-interim euthanasia (week 85-86)
10 Satelite group for recovery (70 week exposure followed by 15 weeks (males) and 16 weeks (females) exposure free)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 3-months study with exposure concentrations of 50, 200 and 1000 ppm.
- Animal assignment: random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- twice daily for morbidity/mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: brief observation before each exposure, detailled observations once weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly intervals until week 28 and every 4 weeks thereafter. Additionally for recovery animals weekly from week 71 to 74 and every 4 weeks thereafter.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 51 and 81 for satelite groups 1 and 2 and week 104 for main group and recovery satelite group
- Blood collected by orbital sinus puncture
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Yes, overnight
- How many animals: 10 from each satelite groups and main group
- Parameters checked:
Haematology: hemoglobin, mean cell volume. red blood cell! count, mean cell hemoglobin. packed cell volume, mean cell hemoglobin concentration, total and differential white blood cell count. and prothrombin time
Clinical chemistry: glutamate oxalale transaminanase. glutamate pyruvate transaminase, alkaline phosphatase, creatinine phosphokinase, sodium, chloride, potasium, calcium, glucose, blood urea nitrogen, albumin, total protein and albumin/globin ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: week 51 and 81 for satelite groups 1 and 2 and week 104 for main group and recovery satelite group.
Immediately after exposure on the day of sampling, the animals were alllowed free acces, to water for a period or approximately 2 hr. Individual
urine samples were then collected overnight (about 12 h) and measured for volume, pH, glucose, bilirubin, urobilinogen, specific gravity, protein, ketone, blood, reducing substances, and microscopic deposits.

Sacrifice and pathology:

Animals were euthanized by an intraperitoneal injection of sodium pentobarbitone and exanguination following an overnight fast.

GROSS PATHOLOGY: Yes
organ weight: adrenals, gonads, kidneys, lungs, spleen, brain, heart, liver and thyroids.

HISTOPATHOLOGY: Yes
Eyes which were fixed in Davidson's fluid, the bone marrow smear was fixed in methanol, testes were fixed in Bouin's solution, all other tissue samples were fixed in 10% neutral buffered formalin.
Adipose tissue, adrenals, aorta, bone marrow smear, brain (medullary, cerebellar, and cortical section), cecum, duodenum, ear canal, esophagus, eyes (and optic nerve), fallopian tubes, femur, ganglia (Iumbar, sacral and dorsal), Harderian glands, heart, hind limbs, (postdistal portions for examitnation of tibial and plantar nerves), ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (mandibular, mediastinal, mesenteric, und bronchial), mammary gland, muscle (quadriceps), nasal turbinates (six levels), ovaries, pancreas, pituitary, prostate, rectum, salivary gland (submaxillary), sciatic nerves, seminal vesicles, skin, spinal cord (high cervical), spleen, stomach (glandular and forestomach), testes with epididymides, thymus (where present), thyroids (with parathyroids where identified), tonge, trachea, urinary bladder, uterus (including cervix), and gross lesions and tissue masses.

Two histopathological evaluations were performed. The first at Hazleton Laboratories, UK. A second, independent. and more detailed review of the respiratory tract tissues was performed at the TNO-ClVO laboratories in the Netherlands.

Statistics:

Body weights and body weight gains, heamatology parameters, measured clinical chemistry parameters, urine volume, terminal body and organ weights, and relative organ weights: One-way analysis of variance followed by pairwise t tests of treated groups against controls.
Urine specific gravity and pH: Kruskal-Wallis test followed by the Wilcoxon rank·sum test.
Survival probabilily functions: Kaplan-Meier technique and compared by the Iog-rank procedure.
Tumor incidence data: evaluated across groups using the Cochran-Armitage trend test.
Nonneoplastic and neoplastic lesions: evaluated between groups using the Fisher's exact test.
Unless otherwise stated, statistical significance was judged at the p=<0.05 level.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Rough haircoat and hunched posture were observed at all concentrations.

Description (incidence):

No consistent increase in mortality associated with treatment was observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reduction in weight gain in 600 ppm groups compared to the controls (10% reduction).
Male rats in the 600 ppm recovery group showed a statistically significant improvement in weight gain. Females did not.

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood glucose levels showed statistically significant decrease in female rats of the 600 ppm group at all sampling points. Considered to be related to reduced food and water consumption .

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in urine volume was observed for all rats of the 600 ppm group at all sampling points and 600 ppm male rats at Week 51. Concomitant effects such as increased specific gravity and decreased pH were noted but were not always statistical significancant.
Considered to be related to reduced food and water consumption.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative lung weights were increased, predominately in females in the 200 and 600 ppm on week 53, onnly on group 600 ppm pn week 83 and in all exposure groupm on week 104. On males weight increaase was only observed on group 600 ppm on week 53.
On the recovery group there were no statistically significant reduction or organ weight after recovery in female rats.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the respiratory epithelium orthe nasal cavity, no nonneoplastic changes observed.
The most prominent a nasal lesion was thinning of the olfactory epithelium accompanied by basal cell hyperplasia. Severe cases: low cuboidal cells lined the thickened submucosa in the olfactory region. Less severe cases: proliferating basal cells were covered by epithelium that resembled respiratory epithelium.
In 600 ppm trated animals, these changes were associated with submucosal edema and with loss of nerve bundles and Bowman's glands or with hyperplasia of glandular structures. As a result infiltrations of inflammalory cells in the epithelium and submucosa as well as leukocytic exudate were those animals.

In 200 ppm groups, the location of the lesions described above were often restricted to, or most pronounced in the anterior part of the dorsal meatus.
ln the 600 ppm concentration group the lesions extended to the posterior part of the olfactory epithelium.
Focal squamous metaplasia of olfactory epithelium (without keratinization) was observed in many 600 ppm-exposed rats and was located mainly on the
top of the dorsal lamellae of the ethmoturbinatcs.
Regeneration of the olfactory epithelium was evident in many rats of the 200 ppm groups and in a few rats of the 600 ppm group. The regenerated epithelium was seen as a layer of stratified undifferentiated epithelium containing small foamy structures resembling nerve bundles and groups of epithelial cells containing yellow-brown pigment resembling acinar cells of the Bowman's glands. The regenerating epithelium was most prominent in the anterior part of the dorsal meatus.
No compound-related nonneoplastic changes were seen in larynx or trachea.
In male and female rats of the 600 ppm groups treatment-related pulmonary changes were observed. Bronchial exfoliation of the lining epithelium was observed in many 600 ppm group males and in a few females. Also, intraluminal fibrosis was observed. This lesion was characterized by fibrous plaques and buds covered by normal bronchial epithelium that projected into the lumen of the airways. An increased incidence of macrophages laden with brown pigment granules located in the main bronchi, in bronchioli, in alveolar spaces. and in the interstitium was observed in the 600 ppm groups.

Nasal and pulmonary lesions similar to those seen in the main study were present in the interim and recovery groups and occurred to about the same incidence and degree as in the main study,with the exeption of bronchial exfoliation that was not observed in any of the interim or recovery group rats.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

12 tumors of the nasal cavity were found. Four of them were classified as benign inverted, endophytic papillomas and were found in the 600 ppm males only; one was classified as a benign exophytic papilloma and was found in one male in the 200 ppm concentration group.
The papillomas were characterized by pseudoacinar structures with cuboidal to columnar cpithelium and. in some cases, muhilayered epithelium with atypical cells and flattening in some areas. The tumors were seen in various part of the nose.
The other tumors all were observed in the 600 ppm group and were classified as malignant carcinoma in situ (one case) or squamous cell carcinomas with varying degrees of keratinization (six cases). The carcinoma in situ was found in a male rat in the olfactory area. One small squamous cell carcinoma was found in another male rat in the ventral floor nomally covered by cuboidal cells. In one male rat a squamous cell carcinoma was noticed in the maxilloturbinate area. A small squamous cell carcinoma occurred in the ethmoturbinate olfactory area in a female rat. Three large squamous cell carcinomas were observed in female
600 ppm rats. These tumors obstructed one side of the nasal cavity and were characterized by invasive growth in nasal bones, soft tissues, and the maxillary sinus. The origin of these tumors could not be established.
In the larynx a squamous cell carcinoma was found in one female rat of the 600 ppm group.
No tracheal or lung tumors were found in the terminal sacrifice animals.
No neoplasms were observed in the 53, 83 week or recovery groups.

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

176 mg/m³ air

Based on:

other: Calculation

Remarks:

Conversion factor: 1ppmv = 3.52 mg/m3 Based on MW of 86.09, 25 °C, 1 atmosphere

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Conclusions:

Inhalation exposure to vapours of vinyl acetate for 104 weeks, lead to non Non-neoplastic lesions in regions of the nasal cavity lined by olfactory epithelium of rats. These effects were statistically significant in the 200 and 600 ppm exposure groups.The NOAEL was determined to be 50 ppm (176 mg/m3) for histopatological effects.
Neoplastic squamous cell carcinomas were observed, mainly on respiratory and olfactory regions in rats exposed to vinyl acetate.The dose-response curve for nasal tumors appears to be nonlinear.

Executive summary:

Chronic toxicity and oncogenicity of vinyl acelate in male and female rats was evaluated in a 104-week study.

Exposure concentrations were 0, 50, 200, and 600 ppm. The study included satelite groups for terminations at approximately 53 and 83 weeks and a recovery group (exposure for 70 weeks and 15-week recovery).

Body weight gain was statistically significant reduced in all 600 ppm groups. Except for female rats of the 600 ppm exposure group, recovery animals showed significant improvements in weight gain relative to controls.

No changes in hematological or clinical chemical parameters were observed that could be unequivocally related to treatment. The only effect noted on clinical chemical parameters were decreases in blood glucose in the 600 ppm females.

There were no adverse effects on survival.

Increases in lung weight were noted in the 600 ppm groups. These changes were associated with bronchial exfoliation, macrophage accumulation, and fibrous plaques and buds extending into the airway lumen, and bronchial/bronchialar epithelial disorganization.

The most significant histopathological changes were noted in the nasal cavity. In the olfactory epithelium, the main non-neoplastic changes included epithelial atrophy, regenerative effects (squamous metaplasia and respiratory metaplasia of olfactory epithelium), basal cell hyperplasia, and epithelial nest-like infolds. No nonneoplastic changes were observed in the respiratory epithelium of rats.

Nonneoplastic changes were similar in the recovery groups.

Oncogenic responses to vinyl acetate exposure were mainly confined to the nasal cavity and included endo- and exophytic papillomas, squamous cell carcinoma, carcinoma in situ in olfactory regions, and endophytic papilloma in respiratory regions. Squamous cell carcinomas were also found either in areas normally covered by cuboidal epithelium or areas of unknown origin.

One squamous cell carcinoma was found in the larynx of a rat of the 600 ppm groups.

The no-observable adverse-effect level for histopathological effects was determined to be 50 ppm (176 mg/m³).

The tumorigenic response appears to be nonlinear.

Reference 4

Endpoint:

carcinogenicity: inhalation

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The substances discussed in this analogue approach are vinyl propionate (target substance) and vinyl acetate (source substance), where both are hypothesized to be readily metabolized [(bio)transformation to a common compound – Scenario 1 of the RAAF guidance] to acetaldehyde (common compound and putative agent) and their corresponding carboxylic acids.
The hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate , leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Vinyl acetate is a high purity substance (99.8% w/w) with a negligible amount of impurities [water (= 0.03 – 0.1%, w/w), acetic acid (= 0.005 – 0.01%, w/w), acetaldehyde (= 0.005 – 0.02%, w/w)] (ECHA 2008). Similarly, vinyl propionate is a high purity substance (99.88%) (Gamer et al. 1997; Intertek 2017), with a negligible amount of impurities [water (= 0.05%)]. The scientific aspects of this Assessment Element have been addressed and the supporting evidence is “acceptable with high confidence”.

3. ANALOGUE APPROACH JUSTIFICATION
The read-across hypothesis and justification establish the structural similarities and differences of the source and target substances. The similarities in their structures and metabolic pathways serve as the foundation for this read-across and justify prediction of vinyl propionate’s toxicological properties from vinyl acetate. The structural differences between the source and target substance are not expected to be linked to differences in toxicological properties. The information reported in Sipi et al. (1992), Chahinian et al. (2002, 2010), the OECD QSAR toolbox’s metabolism/transformation profiler (v4.2, 2018), and well-established theoretical understanding of ester hydrolysis (Adams et al. 2008) provides ample evidence to support acceptability of this Assessment Element. The information provided by these studies also provide strong evidence that the same types of effects are caused in the same biological targets by the common compounds (acetaldehyde and acetic acid). The scientific aspects of this AE have been addressed and the supporting evidence is “acceptable with high confidence”.

The detailed justification for the analogue approach is added to section 13 of this dossier.

4. DATA MATRIX
The detailed data Matrix for the analogue approach is added to section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

other: Read across from analogue Vinyl Acetate

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

205 mg/m³ air

Based on:

other: Read across from analogue Vinyl Acetate

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Remarks on result:

other: Calculated from NOAEL for Vinyl Acetate : 50ppm, Conversion factor: 1ppmv = 4.1 mg/m3 Based on MW of 100.12 (for Vinyl propionate), 25 °C, 1 atmosphere

Conclusions:

Inhalation exposure to vapours of vinyl acetate for 104 weeks, lead to non Nonneoplastic lesions in regions of the nasal cavity lined by olfactory epithelium of rats. These effects were statistically significant in the 200 and 600 ppm exposure groups. The NOAEL was determinet to be 50 ppm (176 mg/m3) for all effects.
Neoplastic squamous cell carcinomas were observed, mainly on respiratory and olfactory regions in rats exposed to vinyl acetate. The dose-response curve for nasal tumors appears to be nonlinear.
The NOAEL for Vinyl Propionate was calculated to be 205 mg/m3 (50 ppm) for all effects.
Based on this read-across, vinyl propionate is classified as carcinogen 2 (H351) according to Regulation (EC) No 1272/2008 (CLP regulation) and no further testing for this endpoint is required.

Executive summary:

Vinyl propionate was assessed based on read-across from vinyl acetate.

As explained in the justification for type of information, the hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate, leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group. The similarities in their structures and metabolic pathways serve as the

foundation for this read-across and justify prediction of vinyl propionate’s toxicological properties from vinyl acetate.

Inhalation exposure to vapours of vinyl acetate for 104 weeks, lead to non Nonneoplastic lesions in regions of the nasal cavity lined by olfactory epithelium of rats. These effects were statistically significant in the 200 and 600 ppm exposure groups. The NOAEL was determined to be 50 ppm (176 mg/m³) for all effects.

Neoplastic squamous cell carcinomas were observed, mainly on respiratory and olfactory regions in rats exposed to vinyl acetate. The dose-response curve for nasal tumors appears to be nonlinear.

The NOAEL for Vinyl Propionate was calculated to be 205 mg/m³ (50 ppm) for all effects.

Based on this read-across, vinyl propionate is classified as carcinogen 2 (H351) according to Regulation (EC) No 1272/2008 (CLP regulation) and no further testing for these endpoint is required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

205 mg/m³

Study duration:

chronic

Species:

other: mouse and rat

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No in-vivo study is available to draw a conclusion on the carcinogenic properties of Vinyl Propionate (CAS 105-38-4).

Data from the analogue vinyl acetate (CAS 108-05-4) were used in a Read-across approach to fulfill the information requirements for this endpoint.

After analyzing the available data for Vinyl Acetate, the RAC report concluded that: “ An increase in tumour incidence has been observed in two species and by two routes of exposure.[...] Extensive mechanistic data suggests that vinyl acetate is carcinogenic by a secondary mechanism with a practical threshold. After inhalation and oral exposure, vinyl acetate is rapidly and effectively hydrolysed to acetic acid and acetaldehyde, the latter is then converted to acetic acid by aldehyde dehydrogenase. The similarities of toxic and carcinogenic effects of vinyl acetate to those of acetaldehyde indicated that acetaldehyde is the critical metabolite that is responsible for the carcinogenic activity of vinyl acetate. [...] The ‘Guidance on the application of the CLP Criteria’ states that the existence of a secondary mechanism of action with the implication of a practical threshold above a certain dose level may lead to a classification in category 2 rather than category 1." (ECHA 2011a, b; see full reference on field additional information)

Therefore, based on the read-across information from the analogue Vinyl acetate (CAS 108-05-4), vinyl propionate (CAS 105-38-4) is classified as carcinogen 2 (H 351) according to Regulation (EC) No 1272/2008 (CLP regulation) and no further testing for this endpoint is required.

Additional information

Data from the analogue vinyl acetate (CAS 108-05-4) were used in a read-across approach to fulfill this endpoint information requirement.

The hypothesis for this analogue approach assumes that vinyl propionate follows the same metabolic pathway established for vinyl acetate, leading to the formation of acetaldehyde. Additional support for the read-across comes from the target and source substance displaying similar physico-chemical and toxicological properties. Vinyl propionate differs structurally from vinyl acetate only in that it poses one additional aliphatic carbon stemming from the ester functional group.

The detailed justification for the analogue approach is added to section 13 of this dossier.

Additional information on analogue vinyl acetate:

In setting vinyl acetate’s harmonized CLP classification (ECHA 2017b), ECHA’s Committee for Risk Assessment (RAC)evaluated the carcinogenic properties of vinyl acetate and concluded that classification as carcinogen 2 (H 351) is warranted. Their rationale is summarized succinctly in the RAC’s Opinion document (ECHA 2011a,b). Key studies used to support the classification are a chronic toxicity and carcinogenicity study in two species of rodents exposed to vinyl acetate via inhalation (Bogdanffy et al. 1994) and a chronic toxicity and carcinogenicity study in two species of rodents administered vinyl acetate in drinking water (Umeda et al. 2004). Specifically, the RAC (2011a,b) reported:

“[E]xtensive mechanistic data suggests that vinyl acetate is carcinogenic by a secondary mechanism with a practical threshold. After inhalation and oral exposure, vinyl acetate is rapidly and effectively hydrolysed to acetic acid and acetaldehyde, the latter is then converted to acetic acid by aldehyde dehydrogenase. The similarities of toxic and carcinogenic effects of vinyl acetate to those of acetaldehyde indicated that acetaldehyde is the critical metabolite that is responsible for the carcinogenic activity of vinyl acetate.”

“Vinyl acetate exhibits local genotoxicity, which again is considered to be caused by the hydrolysis product, acetaldehyde. The effect is thought to have a threshold as the accumulation of acetaldehyde is dependent on the activities of the enzymes involved, i.e. acetaldehyde could accumulate if aldehyde dehydrogenase is saturated.”

For detailed analysis of the available data please refer to:

European Chemicals Agency (ECHA). 2011a. Opinion proposing harmonized classification and labelling at Community level of vinyl acetate. ECHA/RAC/DOC No CLH-O-0000001742-77-01/F. Committee for Risk Assessment (RAC). Adopted June 10.

European Chemicals Agency (ECHA). 2011b. Annex 1, Background document to the Opinion proposing harmonized classification and labelling at Community level of vinyl acetate. ECHA/RAC/DOC No CLH-O-0000001742-77-01/A1. Committee for Risk Assessment (RAC). Adopted June 10.