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Toxicological information

Respiratory sensitisation

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Administrative data

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Jun. 02, 1987 to Dec. 08, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Principles of method if other than guideline:
The animals were initially exposed to the test material by the inhalation route (induction exposure). Following a rest period of 10-14 d during which an immune response may develop, the animals were exposed to a challenge dose (inhalation). The extent and degree of reaction to the challenge exposure in the test animals was compared with that demonstrated by control animals which underwent the same treatment during induction and received the challenge exposure.
GLP compliance:
yes
Remarks:
according to Toxic Substance Control Act (TSCA) GLP

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: 98.5 %
- Impurities (identity and concentrations): 1, 1-dimethyl-m-isopropenyl benzyl isocyanate 3.7 %, acetyl isocyanate 0.35 %, 1, 1-dimethyl-m-isopropenyl benzyl/urethane 0.2 %, m-diisopropenyl benzene 0.05 % and methoxy isocyanate 0.04 %
- Storage condition of test material: Room temperature in an exhaust hood
- Other:
- Boiling point: 302 ° F at 3 mm Hg; 157 °C at 10 mm Hg
- Specific gravity: 1.05
- Vapour pressure: 0.003 mm Hg at 25 °C; 3 mm Hg at 150 °C
- Solubility in water: Reacts slowly
- Flash point: > 200 °F (> 93.3 °C) (Setaflash Closed Cup method)
- Protein conjugate of test material: TMXDI-guinea pig serum albumin conjugate (TMXDI-GPSA) contains 79.4% conjugate and 20.6% buffer salts;
- Lot# : Hazleton Biotechnologies Company Lot# RSL351/013B

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazleton Dutchland, Inc., Denver PA
- Housing: one animal per cage in stainless steel wire-mesh cages (23.5 x 40 x 18 cm)
- Diet: Pellet feed (Pro Lab Guinea Pig Diet, Agway, Inc.Delmont, PA), ad libitum, withheld during exposure
- Water: Tap water (Municipal Authority of Westmoreland Country. Greensburg, PA), ad libitum, withheld during exposure
- Acclimation period: 10 d

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12h dark/12h light
- Temperature: 19.4-21.1 °C (67-70 °F)
- Relative humidity: 48-58%


Test system

Route of induction exposure:
inhalation
Route of challenge exposure:
inhalation
Vehicle:
unchanged (no vehicle)
Concentration:
- Induction phase: 30 µg/L TMXDI aerosol
- Challenge phase: 15-20 µg/L GPSA, followed by 15-20 µg/L aerosol of TMXDI-GPSA
No. of animals per dose:
Test group: 12; Control: 8
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- Exposure period: 3 h
- Test groups: One (twelve animals)
- Control group: One (Eight animals, exposed to filtered air only)
- Frequency of applications: 3h/d for 5 d
- Duration: 5d
- Concentrations: 30 µg/mL TMXDI aerosol
- Induction exposure chamber description:
- Exposure chamber: Stainless steel, with glass windows for observation;
- Volume of the chamber: 900 L
- Air flow rate: 300 L/min
- Induction exposure cage dimensions: 17.5 x 24 x 18 cm wire mesh cages
- Frequency of data recording: Temperature and relative humidity were recorded 6 times/exposure
- Mean daily temperature: test group: 22 °C; control: 24°C



B. CHALLENGE EXPOSURE
- Day(s) of challenge: On Day 22, 23 and 26
- Exposure period: Animals breathed room air for a period of 20 min, followed by 20 min exposure to an aerosol of GPSA and then exposure to TMXDI-GPSA aerosol for a period of 20 min.
- Test groups: One (Twelve animals)
- Control group: One (Eight animals)
- Concentrations: 15-20 µg/L aerosol of GPSA, followed by a 15-20 µg/L aerosol of TMXDI-GPSA after a recovery period of 30 min
- Mean chamber concentration:
- Challenge exposure chamber description:
- Exposure chamber: Four whole body plethysmographs attached to a 2.5 L glass primary chamber
- Air flow rate: 20 L/min
- Acclimatization in chamber: 10-15 min
- Other: A Statham PM 15ETC differential pressure transducer was used to sense pressure changes created during inspiration and expiration of the animals; Frequency of data recording: 15 sec

OTHER:
- Animals assignment: Randomly
- Frequency of observation: Daily
Challenge controls:
None
Positive control substance(s):
none
Negative control substance(s):
none

Results and discussion

Results:
(a) Chamber exposure concentrations:
- The mean induction chamber atmosphere concentration: 31.4±2.78 µg/L (by HPLC) and 30.5±3.15 µg/L (by gravimetric analysis); Mean analytical to nominal concentration ratio ranged between 0.39 -0.49; and mean MMAD of TMXDI aerosol: 1.9 µ with a mean GSD of 2.4.
- Challenge chamber concentration: Mean of individual animals was 17.48±2.21 µg/L for GPSA and 15.04±2.26 µg/L (after adjusting for salt content) for TMXDI-GPSA. The mass median aerodynamic diameter (MMAD) for GPSA representative of exposures was 3.6 µ with GSD of 5.75. (Refer to Table 1 under ‘Attached background material’ for details.)
(b) Clinical observations and mortality:
- Animals exhibited periocular, perinasal, and perioral wetness during and immediately following induction exposures.
- The respiration of these animals decreased and became forced during and following induction exposures. Few animals also displayed audible breathing and/or decreased motor activity during the induction exposure period.
- Mortality: Two animals died during the week of induction exposures (Day 2) and two animals died in the time period following these exposures (Day 19 and 25).
(Refer to Table 2 and 3, under ‘Attached background material’ for details)
(c) Body weights: (Refer to Figure 1 under ‘Attached background material’)
- During the period of the induction exposure, body weight loss was not significant.
- Following induction exposures, body weight gain was noted on Day 12, 19, and 26.
(d) Respiratory rate:
- During the inhalation challenge, none of the animals displayed an increase in respiratory rate greater than 36% of their pre-exposure rate (defined evidence of hypersensitivity. In some cases the pulmonary hypersensitivity criteria were met but they were later found out to be due to animal movements.
(e) Antibody analysis:
- TMXDI-treated guinea pigs had low, but positive titers following the induction exposures.
- Control guinea pigs displayed negative antibody titers.
- The dosing of animals could not be increased in order to increase the antibody titers, since mortality occurred in four out of the twelve exposed animals.
(f) Pathologic evaluation:
- Greater incidence of alveolar histiocytosis was observed in the lungs of the TMXDI-exposed guinea pigs in comparison to control. Further atelectasis was observed in the lungs of both sacrificed and found dead guinea pigs.
- Microscopic examination of the two guinea pigs showed pulmonary edema (one animal), and hyaline membrane formation along with pneumonitis, congestion and hemorrhage. (Refer to Table 4-12 under ‘Attached background material’)
Positive control results:
Not applicable
Negative control results:
Not applicable

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test material was found to be non-sensitising.

Executive summary:

A study was conducted to assess the respiratory sensitization potential of the test substance in guinea pigs, in compliance with GLP. A group of 12 female Hartley guinea pigs were exposed for 5 successive days, 3 h/day by inhalation at a target concentration of 30 µg/L. A second, untreated group of eight animals served as control. The animals were challenged by inhalation during a 20 min exposure on Days 22, 23 and 26 (two weeks following induction exposure) with guinea pig serum albumin (GPSA) and TMXDI-GPSA (after a recovery period of 30 min) at concentrations of 15-20 µg/L. The pulmonary response of the animals to challenge was monitored by recording respiratory rates during and following challenge. Clinical signs of periocular, perioral and perinasal wetness were observed along with respiratory difficulties and diminished motor activity in TMXDI-exposed animals. Four of the twelve TMXDI-exposed animals died during the study. Histopathologic examination of the lungs of TMXDI-exposed animals surviving until the end of the study showed a greater incidence and degree of alveolar histiocytosis than the lungs of control animals. A pulmonary hypersensitivity response was defined as a sustained increase (> 36%) over the mean pre-exposure rate. An immediate pulmonary hypersensitivity response measured in terms of increased respiratory rates was not elicited from any of the guinea pigs upon inhalation challenge. Low, but positive antibody titers for TMXDI were observed in exposed guinea pigs. Hence, under the study conditions, the test substance was found to be non-sensitising (Burleigh-Flayer, 1988).