Butyl chloroformate

Endpoint summary

Description of key information

In a 28 day inhalation repeated dose toxicity study (OECD 412, GLP) the NOAEC was determined to be 28.2 (highest dose tested) and 10.0 µg/L, for systemic and local effects respectively.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant non-guideline, available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Portage, Shaver Road, Portage, Michigan, U.S.A.
- Age at study initiation: 7-8 weeks
- Weight at study initiation (group mean bodyweights): Males 154.8 - 156.2 g, Females 130.4 - 131.2 g
- Housing: 5 animals of the same sex to a cage, in suspended polypropylene cages fitted with stainless steel mesh tops and floors. Each cage measured 56 cm long, 38 cm wide and 18 cm high.
- Diet: laboratory rat food (Biosure Diet LAD 1, Biosure, Lavender Mill, Manea, Cambridgeshire, England), ad libitum
- Water: Ad libitum
- Acclimation period: Approx. 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.0 - 21.5
- Humidity (%): 40 - 68
- Air changes: Each group of rats was kept in a separate ventilated cabinet to prevent any possible cross-contamination between groups once exposures had commenced. The ventilated cabinets drew their air supply from the holding room.
- Photoperiod (hrs dark / hrs light): 12 hours light (0800 -2000 hours) and 12 hours dark per 24 hours.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- The vapour of the test substance was generated by metering the test substance from an all polypropylene disposable syringe (Sigma Chemical Co.) mounted on an infusion pump (Precidor 5 type 5003) to a sintered glass frit, contained in a glass vessel. Air was fed through this vessel at a rate of 5 L/minute and was heated by passage through a coiled copper tube immersed in a water bath maintained at 80°C. The vapour produced passed from the all glass vaporiser into the base of a glass elutriation column where it was mixed with diluent air entering the column at a rate of 145 litres per minute. The test atmosphere then passed into the inlet duct of the exposure chamber. Different vapour concentrations appropriate to each exposure group were achieved by varying the rate at which the test substance was metered to the glass frit.
- The exposure chambers were constructed of stainless steel and glass. Each of the 4 chambers used was approximately 0.7 m3 in volume. The chambers were of square plan section fitted with a pyramidal base and top. A square-shaped 3 inch diameter tubular perforated exhaust plenum was fitted in the base of each chamber below exposure cage level. A perforated dispersion plate was fitted at the point of air entry. A 1.5 inch drainage duct fitted with a ball valve was present in the centre base of the chamber and was connected with a drainage system. The chamber air flow was 150 litres per minute. A magnehelic pressure gauge (0 - 100 mm water gauge) was connected with each chamber by a nylon tube. A separate exposure chamber was used for each group of rats. The air control animals were exposed using a similar system to that used for the test groups receiving test substance vapour.
- Temperature and humidity in air chamber: 21.2 - 22.3°C and 34 - 49%, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

-The concentration of the test substance present in the exposure chambers used for Groups 2, 3 and 4 was determined on at least 3 occasions during each exposure. Samples were taken at approximately 1, 3 and 5 hours from the start of exposure.
- Samples of test atmosphere were withdrawn at 2 litres per minute through charcoal adsorption tubes. The contents of the tubes were eluted into accurately measured 5 mL aliquots of carbon disuiphide.
- The samples were analysed by flame ionisation chromatography using external standards.

Duration of treatment / exposure:

28 days (6 hours per day)

Frequency of treatment:

5 days a week

Remarks:

Doses / Concentrations:
2.8, 10 , 28.2 µg/L
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CLINICAL SIGNS
- During exposure: Clinical signs during exposure were recorded as a group response where all visible animals appeared to be responding similarly or a proportion were affected.
- At other times: Animals were examined twice each day, usually prior to loading and immediately following unloading from the chambers on exposure days, and in the morning and afternoon of non-exposure days.

BODY WEIGHT:
- Each rat was weighed for allocation to groups, then at weekly intervals, commencing 1 week before the start of dosing and continuing throughout the study. In addition, the weight of each rat at necropsy was recorded.

FOOD CONSUMPTION:
- The quantity of food consumed by each cage of rats was recorded weekly commencing one week prior to the start of exposures until the end of the study.

HAEMATOLOGY
- Samples of blood were withdrawn from the orbital sinus of all rats during Week 4 of the study . The samples were removed while the rats were lightly anaesthetised with ether. All rats were deprived of food overnight prior to removal of blood. Water was available during this overnight period. EDTA and sodium citrate (thrombotest) anticoagulants were used. The parameters measured were as follows: Packed cell volume, Haemoglobin, Red cell count, Mean corpuscular haemoglobin concentration, Mean corpuscular volume, Mean corpuscular haemoglobin, Total white cell count, Platelets, Differential count, Reticulocyte count, Thrombotest

BLOOD BIOCHEMISTRY
-The blood was placed into proprietary blood collection vials containing lithium heparin anticoagulant. The vials were centrifuged at 3200 g for 3 minutes and the plasma analysed for the appropriate parameters: Glucose, Glutamic-pyruvic transaminase, Glutamic-oxaloacetic transaminase, Total protein, Albumin, Globulin, Albumin/Globulin ratio, Urea nitrogen, Alkaline phosphatase, Total bilirubin, Creatinine, Sodium (Na), Potassium (K), Calcium (Ca), Inorganic phosphorus (P), Chloride (CL) , Cholesterol

Sacrifice and pathology:

MACROSCOPIC PATHOLOGY AND ORGAN WEIGHT ANALYSIS
- At necropsy a detailed macroscopic examination of all rats was performed. The following organs were dissected from each rat and weighed: Brain, Liver, Testes with epididymides, Pituitary, Kidneys, Ovaries, Lungs, Adrenals.

MICROSCOPIC EXAMINATION
- Light microscopic exanimation was performed on 4 µm thick sections stained with haematoxylin and eosin. The following tissues were examined from all rats: Adrenals, Kidneys, Spleen, Gross abnormalities, Liver, Trachea, Nasal passages, Lungs, Larynx, and Heart.

Statistics:

All statistical analyses were carried out separately for males and females. For all parameters the analyses were carried out using the individual animal as the basic experimental unit. Food consumption data were not statistically analysed as only a single cage/group/sex was present. Bodyweight data were analysed using weight gains. The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data: If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%) the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise: (1) Bartlett's test was applied to test for heterogeneity of variance between treatments; where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained. (2) If no significant heterogeneity was detected (or if a satisfactory transformation was found, a one-way analysis of variance was carried out. If significant heterogeneity-of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used. (3) Except for pre-exposure data, analyses of variance were followed by Student's t-test and Williams' test for a dose-related response, although only Williams' test was reported. The Kruskal-Wallis analyses were followed by Shirley's test, the non-parametric equivalent of the 't' and Williams' tests. Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, the final bodyweight was used as covariate in an attempt to allow for differences in bodyweight which might affect the organ weights.

Details on results:

CLINICAL SIGNS AND MORTALITY
- No mortaility occurred
- During exposure pilo-erection was noted in Group 4 (High dose)

BODY WEIGHT AND WEIGHT GAIN:
- Minimal differences in bodyweight gain between exposed and control groups were considered probably not of toxicological significance.

FOOD CONSUMPTION
- Food consumption for female rats from Group 2 (Low dose) and male and female rats from Groups 3 (Intermediate dose) and 4 was marginally lower than control values. The differences were very small and were probably not treatment-related.

HAEMATOLOGY
- The following statistically significant differences compared with control values were seen: Total numbers of white blood cells increased in male rats from Groups 3 and 4. Neutrophil numbers increased in male rats from Group 4. The increase in numbers was not dose-related and no concomitant increase was seen in female rats. The differences are considered unlikely to be of any toxicological significance.

BIOCHEMISTRY
- The following statistically significant differences compared with control values were noted: Reduced albumin concentration in male rats from Group 4. Increased globulin concentration in male rats from Groups 3 and 4. Increased A/G ratio in male rats from Group 4. Reduced urea nitrogen in male rats from Groups 3 and 4. These differences were minimal. Differences in A/G ratio and urea nitrogen were not dose-related and no statistically significant differences were seen in female rats. They are therefore considered not to be of toxicological significance.

ORGAN WEIGHTS
- Increased lung weights were recorded for male and female rats in Group 4. The difference was statistically significant for control values for male rats only (P<0.01). The increased lung weights were considered to be related to treatment.

MACROSCOPIC PATHOLOGY
- No changes were seen that were considered to be of toxicological significance.

MICROSCOPIC PATHOLOGY
- Treatment related changes were confined to the carina: these were confined to rats in Group 4 (High dose) and included minimal focal epithelial hyperplasia in 1/5 male and 3/5 female rats together with minimal focal crowding of epithelial cells in 3/5 male rats

Dose descriptor:

NOAEC

Remarks:

Local

Effect level:

10 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on lung weight en carina observed at the highest dose tested.

Dose descriptor:

NOEC

Remarks:

Systemic

Effect level:

28.2 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic toxicological relevant effects observed.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

28.2 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant non-guideline, available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Portage, Shaver Road, Portage, Michigan, U.S.A.
- Age at study initiation: 7-8 weeks
- Weight at study initiation (group mean bodyweights): Males 154.8 - 156.2 g, Females 130.4 - 131.2 g
- Housing: 5 animals of the same sex to a cage, in suspended polypropylene cages fitted with stainless steel mesh tops and floors. Each cage measured 56 cm long, 38 cm wide and 18 cm high.
- Diet: laboratory rat food (Biosure Diet LAD 1, Biosure, Lavender Mill, Manea, Cambridgeshire, England), ad libitum
- Water: Ad libitum
- Acclimation period: Approx. 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.0 - 21.5
- Humidity (%): 40 - 68
- Air changes: Each group of rats was kept in a separate ventilated cabinet to prevent any possible cross-contamination between groups once exposures had commenced. The ventilated cabinets drew their air supply from the holding room.
- Photoperiod (hrs dark / hrs light): 12 hours light (0800 -2000 hours) and 12 hours dark per 24 hours.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- The vapour of the test substance was generated by metering the test substance from an all polypropylene disposable syringe (Sigma Chemical Co.) mounted on an infusion pump (Precidor 5 type 5003) to a sintered glass frit, contained in a glass vessel. Air was fed through this vessel at a rate of 5 L/minute and was heated by passage through a coiled copper tube immersed in a water bath maintained at 80°C. The vapour produced passed from the all glass vaporiser into the base of a glass elutriation column where it was mixed with diluent air entering the column at a rate of 145 litres per minute. The test atmosphere then passed into the inlet duct of the exposure chamber. Different vapour concentrations appropriate to each exposure group were achieved by varying the rate at which the test substance was metered to the glass frit.
- The exposure chambers were constructed of stainless steel and glass. Each of the 4 chambers used was approximately 0.7 m3 in volume. The chambers were of square plan section fitted with a pyramidal base and top. A square-shaped 3 inch diameter tubular perforated exhaust plenum was fitted in the base of each chamber below exposure cage level. A perforated dispersion plate was fitted at the point of air entry. A 1.5 inch drainage duct fitted with a ball valve was present in the centre base of the chamber and was connected with a drainage system. The chamber air flow was 150 litres per minute. A magnehelic pressure gauge (0 - 100 mm water gauge) was connected with each chamber by a nylon tube. A separate exposure chamber was used for each group of rats. The air control animals were exposed using a similar system to that used for the test groups receiving test substance vapour.
- Temperature and humidity in air chamber: 21.2 - 22.3°C and 34 - 49%, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

-The concentration of the test substance present in the exposure chambers used for Groups 2, 3 and 4 was determined on at least 3 occasions during each exposure. Samples were taken at approximately 1, 3 and 5 hours from the start of exposure.
- Samples of test atmosphere were withdrawn at 2 litres per minute through charcoal adsorption tubes. The contents of the tubes were eluted into accurately measured 5 mL aliquots of carbon disuiphide.
- The samples were analysed by flame ionisation chromatography using external standards.

Duration of treatment / exposure:

28 days (6 hours per day)

Frequency of treatment:

5 days a week

Remarks:

Doses / Concentrations:
2.8, 10 , 28.2 µg/L
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CLINICAL SIGNS
- During exposure: Clinical signs during exposure were recorded as a group response where all visible animals appeared to be responding similarly or a proportion were affected.
- At other times: Animals were examined twice each day, usually prior to loading and immediately following unloading from the chambers on exposure days, and in the morning and afternoon of non-exposure days.

BODY WEIGHT:
- Each rat was weighed for allocation to groups, then at weekly intervals, commencing 1 week before the start of dosing and continuing throughout the study. In addition, the weight of each rat at necropsy was recorded.

FOOD CONSUMPTION:
- The quantity of food consumed by each cage of rats was recorded weekly commencing one week prior to the start of exposures until the end of the study.

HAEMATOLOGY
- Samples of blood were withdrawn from the orbital sinus of all rats during Week 4 of the study . The samples were removed while the rats were lightly anaesthetised with ether. All rats were deprived of food overnight prior to removal of blood. Water was available during this overnight period. EDTA and sodium citrate (thrombotest) anticoagulants were used. The parameters measured were as follows: Packed cell volume, Haemoglobin, Red cell count, Mean corpuscular haemoglobin concentration, Mean corpuscular volume, Mean corpuscular haemoglobin, Total white cell count, Platelets, Differential count, Reticulocyte count, Thrombotest

BLOOD BIOCHEMISTRY
-The blood was placed into proprietary blood collection vials containing lithium heparin anticoagulant. The vials were centrifuged at 3200 g for 3 minutes and the plasma analysed for the appropriate parameters: Glucose, Glutamic-pyruvic transaminase, Glutamic-oxaloacetic transaminase, Total protein, Albumin, Globulin, Albumin/Globulin ratio, Urea nitrogen, Alkaline phosphatase, Total bilirubin, Creatinine, Sodium (Na), Potassium (K), Calcium (Ca), Inorganic phosphorus (P), Chloride (CL) , Cholesterol

Sacrifice and pathology:

MACROSCOPIC PATHOLOGY AND ORGAN WEIGHT ANALYSIS
- At necropsy a detailed macroscopic examination of all rats was performed. The following organs were dissected from each rat and weighed: Brain, Liver, Testes with epididymides, Pituitary, Kidneys, Ovaries, Lungs, Adrenals.

MICROSCOPIC EXAMINATION
- Light microscopic exanimation was performed on 4 µm thick sections stained with haematoxylin and eosin. The following tissues were examined from all rats: Adrenals, Kidneys, Spleen, Gross abnormalities, Liver, Trachea, Nasal passages, Lungs, Larynx, and Heart.

Statistics:

All statistical analyses were carried out separately for males and females. For all parameters the analyses were carried out using the individual animal as the basic experimental unit. Food consumption data were not statistically analysed as only a single cage/group/sex was present. Bodyweight data were analysed using weight gains. The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data: If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%) the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise: (1) Bartlett's test was applied to test for heterogeneity of variance between treatments; where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained. (2) If no significant heterogeneity was detected (or if a satisfactory transformation was found, a one-way analysis of variance was carried out. If significant heterogeneity-of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used. (3) Except for pre-exposure data, analyses of variance were followed by Student's t-test and Williams' test for a dose-related response, although only Williams' test was reported. The Kruskal-Wallis analyses were followed by Shirley's test, the non-parametric equivalent of the 't' and Williams' tests. Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, the final bodyweight was used as covariate in an attempt to allow for differences in bodyweight which might affect the organ weights.

Details on results:

CLINICAL SIGNS AND MORTALITY
- No mortaility occurred
- During exposure pilo-erection was noted in Group 4 (High dose)

BODY WEIGHT AND WEIGHT GAIN:
- Minimal differences in bodyweight gain between exposed and control groups were considered probably not of toxicological significance.

FOOD CONSUMPTION
- Food consumption for female rats from Group 2 (Low dose) and male and female rats from Groups 3 (Intermediate dose) and 4 was marginally lower than control values. The differences were very small and were probably not treatment-related.

HAEMATOLOGY
- The following statistically significant differences compared with control values were seen: Total numbers of white blood cells increased in male rats from Groups 3 and 4. Neutrophil numbers increased in male rats from Group 4. The increase in numbers was not dose-related and no concomitant increase was seen in female rats. The differences are considered unlikely to be of any toxicological significance.

BIOCHEMISTRY
- The following statistically significant differences compared with control values were noted: Reduced albumin concentration in male rats from Group 4. Increased globulin concentration in male rats from Groups 3 and 4. Increased A/G ratio in male rats from Group 4. Reduced urea nitrogen in male rats from Groups 3 and 4. These differences were minimal. Differences in A/G ratio and urea nitrogen were not dose-related and no statistically significant differences were seen in female rats. They are therefore considered not to be of toxicological significance.

ORGAN WEIGHTS
- Increased lung weights were recorded for male and female rats in Group 4. The difference was statistically significant for control values for male rats only (P<0.01). The increased lung weights were considered to be related to treatment.

MACROSCOPIC PATHOLOGY
- No changes were seen that were considered to be of toxicological significance.

MICROSCOPIC PATHOLOGY
- Treatment related changes were confined to the carina: these were confined to rats in Group 4 (High dose) and included minimal focal epithelial hyperplasia in 1/5 male and 3/5 female rats together with minimal focal crowding of epithelial cells in 3/5 male rats

Dose descriptor:

NOAEC

Remarks:

Local

Effect level:

10 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on lung weight en carina observed at the highest dose tested.

Dose descriptor:

NOEC

Remarks:

Systemic

Effect level:

28.2 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic toxicological relevant effects observed.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

10 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a GLP compliant inhalation study performed according to OECD guideline 412, five SD rats per sex per dose were exposed to the vapour of the test substance by whole-body exposure for 6 hours a day, 5 days a week over a period of 28 days. Control rats were similarly treated but no substance was introduced into the exposure chamber. Average tested concentrations were 2.8, 10 and 28.2 µg/L. Animals were examined for mortality, clinical signs, bodyweight, food consumption, haematology, biochemistry, organ weights macroscopic pathology and microscopic pathology. No adverse effects of exposure were seen in rats exposed for 28 days to the test substance at average concentrations of 2.8 and 10.0 µg/L. In the animals exposed to 28.2 µg/L, increased lung weights were recorded for male and female rats. The difference was statistically significant for control values for male rats only. In addition, treatment related changes were observed in the carina. They included minimal focal epithelial hyperplasia in 1/5 male and 3/5 female rats together with minimal focal crowding of epithelial cells in 3/5 male rats. The NOAEC for this study was therefore considered to be 28.2 and 10.0 µg/L, for systemic and local effects respectively.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
One repeated dose inhalation toxicity study is available.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
One repeated dose inhalation toxicity study is available.

Justification for classification or non-classification

Based on the results obtained in this study, classification for repeated dose toxicity in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 is not warranted.

According to the CLP (EC/1272/2008), Annex I: 3.9.1.1. specific toxic effects that are specifically addressed at the other toxicological endpoints (acute toxicity, corrosivity, etc.) are not included to classification for STOT RE. The observed effects on the carina can be referred to the corrosivity of the substance and therefore, a classification for STOT RE is not justified.