Registration Dossier

Administrative data

Description of key information

Oral studies: 14 day and 28 day repeated dose oral gavage studies and an oral gavage OECD 421 reproductive/developmental screening study - all in rats
Inhalation studies: 14 day range finding inhalation study in rats and a 13-week inhalation study in rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rangefinder to set dose levels for OECD 421
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
62.5 mg/mL
Basis:
actual ingested
Remarks:
Doses / Concentrations:
125 mg/mL
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/mL
Basis:
actual ingested
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Dose descriptor:
dose level:
Effect level:
> 250 - < 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The results of this range-finding study included body weight effects as well as point of contact irritation in the stomach at 500 mg/kg bwt/day, and death and moribundity at 1000 mg/kg bwt/day.
Critical effects observed:
not specified

Table 1. Summary of Clinical Signs - Males

 Dose (mg/kg bwt/day)  0  250  500  1000
 Found Dead        
  Number of Observations  -  -  -  1
  Number of Animals  -  -  -  1
  Days from - to  -  -  -  7 - 7
 Gasping        
  Number of Observations  -  -  -  1
  Number of Animals  -  -  -  1
  Days from - to  -  -  -  9 - 9
 Killed -moribund        
  Number of Observations  -  -  -  1
  Number of Animals  -  -  -  1
  Days from - to  -  -  -  9 - 9
 Salivation        
  Number of Observations  -  -  9  21
  Number of Animals  -  -  3  3
  Days from - to  -  -  8 - 13  2 - 14
 Lateral Recumbent        
  Number of Observations  -  -  -  1
  Number of Animals  -  -  -  1
  Days from - to  -  -  -  9 - 9
 Hypo Activity        
  Number of Observations  -  -  5  24
  Number of Animals  -  -  2  3
  Days from - to  -  -  7 - 13  2 - 14

Table 2. Summary of Clinical Signs - Females       

 Dose (mg/kg bwt/day)  0  250  500  1000
 Found Dead        
  Number of Observations  -  -  -  1
  Number of Animals  -  -  -  1
  Days from - to  -  -  -  9 - 9
 Gasping        
  Number of Observations  -  -  -  1
  Number of Animals  -  -  -  1
  Days from - to  -  -  -  9 - 9
 Salivation        
  Number of Observations  -  -  3  22
  Number of Animals  -  -  2  3
  Days from - to  -  -  8 - 14  3 - 14
 Hypo Activity        
  Number of Observations  -  -  5  31
  Number of Animals  -  -  3  3
  Days from - to  -  -  8 - 14  2 - 14

Table 3. Summary of Body Weights (g) - Males       

                 Days
 Dose (mg/kg bwt/day)    1  4  7  11  14
 0  Mean  216.963  230.943  247.083  267.630  282.007
   S.D.  14.408  16.893  16.764  19.918  19.566
   N  3  3  3  3  3
 250  Mean  220.237  232.433  247.930  266.290  278.890
   S.D.  15.033  16.246  18.519  21.207  25.148
   N  3  3  3  3  3
 500  Mean  220.557  225.433  236.367  254.317  266.123
   S.D.  14.340  17.421  25.585  29.380  32.655
   N  3  3  3  3  3
 1000  Mean  224.503  223.053  219.235  225.030  234.000
   S.D.  13.078  12.516  3.528  -  -
   N  3  3  2  1  1

Table 4. Summary of Body Weights - Females       

                 Days
 Dose (mg/kd bwt/day)    1  4  7  11  14
 0  Mean  153.277  159.123  163.733  170.860  177.957
   S.D.  9.566  8.977  6.744  6.937  6.322
   N  3  3  3  3  3
 250  Mean  148.653  153.420  160.680  170.790  180.883
   S.D.  10.387  10.621  9.020  8.868  3.986
   N  3  3  3  3  3
 500  Mean  155.333  158.100  164.513  169.550  175.537
   S.D.  10.842  9.824  12.273  14.872  12.797
   N  3  3  3  3  3
 1000  Mean  155.153  155.883  155.633  162.440  166.685
   S.D.  4.377  11.942  15.280  32.654  35.405
   N  3  3  3  2  2

Table 5. Summary of Net Body Weight Gains (g) - Males       

                 Days  Abs Gain  % Gain
 Dose (mg/kg bwt/day)  1 -4  4 -7  7 -11  11 -14  1 -14  1 -14
 0  Mean  13.980  16.140  20.547  14.377  65.043  29.96
   S.D.  2.653  1.360  3.155  2.723  5.831  1.51
   N  3  3  3  3  3  3
 250  Mean  12.197  15.497  18.360  12.600  58.653  26.51
   S.D.  1.896  2.296  2.793  4.100  10.191  2.75
   N  3  3  3  3  3  3
 500  Mean  4.877*  10.933  17.950  11.807  45.567  20.37
   S.D.  4.206  8.760  4.200  6.496  18.900  7.35
   N  3  3  3  3  3  3
 1000  Mean  -1.450*  -8.050  3.300*  8.970  16.460  7.57
   S.D.  0.928  17.876  -  -  -  -
   N 3  2  1  1  1  1

* Significantly lower than the vehicle control group

Table 6. Summary of of Feed Intake (g/rat/day) - Males

              Days
 Dose (mg/kg bwt/day)    1 -4  4 -7  7 -11  11 -14
 0  Mean  18.533  19.992  20.809  20.599
   S.D.  2.473  1.693  1.251  1.484
   N  3  3  3  3
 250  Mean  18.464  20.336  20.951  20.574
   S.D.  1.322  1.304  1.665  2.160
   N  3  3  3  3
 500  Mean  15.821  19.074  19.777  20.190
   S.D.  4.528  3.541  5.086  2.618
   N  3  3  3  3
 1000  Mean  12.788  11.782*  11.160  15.973
   S.D.  1.870  1.803  -  -
   N  3  2  1  1

* Significantly lower than the control group

Table 7. Summary of Terminal Fasting Body Weights and Organ to Body Weight Ratios - Femal

Dose (mg/kg bwt/day)
   Terminal Fasting BW (g)  Kidneys (g)  Liver (g)  Kidneys (Percentage)  Liver (Percentage)
0  Mean  170.06  1.2352  5.2234  0.726  3.074
   SD  8.11  0.0752  0.1225  0.032  0.103
   N  3  3  3  3  3
 250  Mean  170.83  1.3038  5.9431  0.763  3.480
   SD  4.76  0.0528  0.4316  0.010  0.262
   N  3  3  3  3  3
 500  Mean  168.87  1.3602  6.0324  0.805  3.575
   SD  15.20  0.1765  0.5874  0.058  0.213
   N  3  3  3  3  3
 1000  Mean  158.48  1.2118  7.3775*  0.772  4.785*
   SD  34.83  0.1669  0.2055  0.064  1.181
   N  2  2  2  2  2
   % Diff  --  --  41  --  56

*Significantly higher than the vehicle control group

-- Not calculated as difference is not statistically significant

Table 8. Summary of Gross Pathological Findings

            Males                    Females
 Dose (mg/kg bwt/day)  0  250  500  1000  0  250  500  1000
 Number of Animals on Study  3  3  3  3  3  3  3  3
 Number of Animals Completed  (3)  (3)  (3)  (3)  (3)  (3)  (3)  (3)
 DUODENUM;                
  Submitted  (3)  (3)  (3)  (3)  (3)  (3)  (3)  (3)
  No Visible Lesions  3  3  3  2  3  3  3  3
  Erosion; mucosal; multifocal  0  0  0  1  0  0  0  0
 EYE;                
  Submitted  (3)  (3)  (3)  (3)  (3)  (3)  (3)  (3)
  No Visible Lesions  3  3  3  3  3  3  3  2
  Opaque; unilateral  0  0  0  0  0  0  0  1
 ILEUM WITH PEYERS PATCH;                
  Submitted  (3)  (3)  (3)  (3)  (3)  (3)  (3)  (3)
  No Visible Lesions  3  3  3  2  3  3  3  3
  Erosion; mucosal; multifocal  0  0  0  1  0  0  0  0
 JEJUNUM;                
  Submitted  (3)  (3)  (3)  (3)  (3)  (3)  (3)  (3)
  No Visible Lesions  3  3  3  2  3  3  3  2
  Thickened; multifocal  0  0  0  0  0  0  0  1
  Erosion; mucosal; multifocal  0  0  0  1  0  0  0  0
 STOMACH;                
  Submitted  (3)  (3)  (3)  (3)  (3)  (3)  (3)  (3)
  No Visible Lesions  3  3  0  1  3  3  1  0
  Focus (i); raised; non-glandular mucosa  0  0  3  0  0  0  2  1
  Thickened; non-glandular mucosa; diffuse  0  0  0  1  0  0  0  0
  Discoloration; red; glandular mucosa; multifocal 0  0  0  1  0  0  0  1
  Erosion; non-glandular mucosa; multifocal  0  0  0  1  0  0  0  2
 TRACHEA;                
  Submitted  (3)  (3)  (3)  (3)  (3)  (3)  (3)  (3)
  No Visible Lesions  3  3  3  3  3  3  3  2
  Frothy contents  0  0  0  0  0  0  0  1
 INTESTINE;                
  Submitted  (3)  (3)  (3)  (3)  (3)  (3)  (3)  (3)
  No Visible Lesions  3  3  3  2  3  3  3  3
  Contents discoloured-brown  0  0  0  1  0  0  0  0

Note: All other tissues were examined and no gross abnormalities were observed for all the animals.

Conclusions:
The results of this range-finding study included body weight effects as well as point of contact irritation in the stomach at 500 mg/kg bwt/day, and death and moribundity at 1000 mg/kg bwt/day. Since the purpose of this study was to assist in determining dose levels for an upcoming definitive study, 500 and 1000 mg/kg bwt/day are both judged to be too high of a dose for the further screening test on Reproduction/Developmental toxicity (OECD 421), due to the longer duration of exposure, as well as the additional stressors of gestation and lactation phases on the dams.
Executive summary:

This repeated dose toxicity study was conducted to evaluate the toxicity potential of the test substance, 1,3 and 1,4 Cyclohexanecarboxaldehyde, Lot Number 201001644 2nd pass Bottle #2, when administered orally by gavage to Wistar rats for 14 consecutive days and in order to select dose levels for a further screening test on Reproduction/Developmental toxicity.

The test substance was administered at an equivolume of 4 mL/kg/day to G2 (low dose), G3 (mid dose) and G4 (high dose) groups of rats at the dose levels of 250, 500 and 1000 mg/kg body weight/day, respectively. Concurrently, vehicle control group (G1) animals were administered the vehicle alone (corn oil) at the same dose volume. Each group in the study was comprised of 3 rats per sex.

The dose formulations were analyzed for homogeneity and active ingredient concentration twice during the treatment period. The formulations were found to be homogeneous and the analyzed concentrations were well within the acceptance criteria of ± 15% of the theoretical concentrations. No evidence of the active ingredient was detected in the control samples at the established lowest calibration standard concentration of 53.8 μg/ml, carried out under Advinus Study No. G8236.

All the rats in the study were observed for clinical signs twice on the first day of treatment. Subsequently, the observation was carried out thrice daily until the end of treatment. The rats were observed for morbidity and mortality twice daily (once daily on weekends). Individual body weights were recorded prior to test substance administration for all rats on Day 1 and on Days 4, 7, 11 and 14. Additionally, body weight was also measured on Day 15 following overnight fasting. Feed consumption

was measured on Day 4 (representing the feed consumption between Days 1-4) and on Days 7, 11 and 14 (representing the feed consumption between Days 4-7, Days 7-11 and Days 11-14, respectively) for each rat. Kidney and liver weights were recorded from all the terminally sacrificed rats after gross pathology examination.

The daily oral gavage of 1,3 and 1,4 Cyclohexanecarboxaldehyde for 14 consecutive days at the dose of 250 mg/kg body weight/day had no test substance-related effects on the general health of the animals, body weights, net body weight gains, feed consumption, organ weights of the liver and kidneys and gross pathology. However, at 1000 mg/kg bwt/day, treatment related mortalities were observed (2/6), and one male rat was euthanized moribund. Clinical signs of hypo activity and salivation were observed at 500 and 1000 mg/kg bwt/day. The mean and net body weight gains and feed consumption were lower at 1000 mg/kg bwt/day compared to the concurrent control group. Percent body weight gain was also decreased in males (32%) and females (20%) at 500 mg/kg bwt/day. The treatment resulted in an increase in the absolute and relative liver weights in females at 1000 mg/kg bwt/day. There was also a marginal increase in absolute (15%) and relative (16%) liver weights in females at 500 mg/kg bwt/day. Gross pathological findings included erosions and thickening of the stomach and intestines of some animals in the 1000 mg/kg bwt/day group. Raised foci were present in the stomach of 3 males and 2 females in the 500 mg/kg bwt/day group.

The results of this range-finding study included body weight effects as well as point of contact irritation in the stomach at 500 mg/kg bwt/day, and death and moribundity at 1000 mg/kg bwt/day. Since the purpose of this study was to assist in determining dose levels for an upcoming definitive study, 500 and 1000 mg/kg bwt/day are both judged to be too high of a dose for the further screening test on Reproduction/Developmental toxicity (OECD 421), due to the longer duration of exposure, as well as the additional stressors of gestation and lactation phases on the dams.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Two repeated dose oral studies in rats are available for this material, a 14-day repeated dose study performed as a range finder for the OECD 421 and a 28-day repeated dose oral study. Taken together these three studies indicate that the test material does not cause systemic toxicity following repeated dose exposure. In addition, an OECD 421 reproductive screening study is available and provides additional information that supports the
guideline repeated dose toxicity studies.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 July 2013-6 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was not conducted according to guidelines but was conducted according to GLPs and the report contains sufficient data for interpretation of study results.
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Qualifier:
according to
Guideline:
other: EEC, Part B.29
Principles of method if other than guideline:
This study was also designed to evaluate the potential for local (portal-of-entry) and systemic toxicity from inhalation of 1,3- and 1,4-cyclohexanedicarboxaldehyde.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: see "Details on inhalation exposure"
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
six hours/day, five consecutive days/week for 13 weeks.
No. of animals per sex per dose:
Male and female (each): 10 animals at 0 mg/m3
Male and female (each): 10 animals at 0.3 mg/m3
Male and female (each): 10animals at 2.67 mg/m3
Male and female (each): 10animals at 24.0 mg/m3
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
local signs of irritation in the respiratory tract
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEC
Remarks:
Systemic
Effect level:
> 24 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; mortality; body weight; food consumption; food efficiency; water consumption and compound intake; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology; other.
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
ca. 2.67 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based upon the localized, point of contact histopathological effects in the anterior nasal passage.
Dose descriptor:
NOEC
Remarks:
Local
Effect level:
ca. 2.67 mg/m³ air
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based upon the localized, point of contact histopathological effects in the anterior nasal passage.
Critical effects observed:
not specified

There were no exposure-related effects on body weights/body weight gains, feed consumption, clinical observations, functional tests,hematology, prothrombin time, clinical chemistry, urinalysis, or organ weights. Based on BAL analyses, male and female rats exposed to1,3- and 1,4-cyclohexanedicarboxaldehyde vaporsat concentrations up to and including 24.0 mg/m3showed no evidence of exposure-related cellular injury or inflammation. Compared to control rats, no increase in total BAL cell numbers and no evidence of acute neutrophilic inflammation were detected in any test material-exposed group. No treatment-related alterations were noted in BAL protein or LDH levels.

There were no treatment-related gross pathologic observations.

There was no histopathological evidence of treatment-related systemic toxicity in males or females exposed to the highest exposure concentration of 24.0 mg/m3. Males exposed to 2.67 or 24.0 mg/m3and females exposed to 24.0 mg/m3had treatment-related histopathological changes that were confined to the anterior nasal cavity consistent withlocalized, point of contact irritant effects resulting from repeated inhalation of 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors. The effects observed in the males at the 2.67 mg/m3 were very slight and represented an adaptive change (increase in mucouse secretion) rather than an adverse effect. Nasal airways of males exposed to 0.30 mg/m3and females exposed to 0.30 or 2.67 mg/m3had no treatment-related changes. There were no treatment-related histopathological changes in the posterior nasal airways, larynx, trachea or lungs in males or females in any of the exposure groups.

A limited assessment of the effects of repeated inhalation exposure to 1,3- and 1,4-cyclohexanedicarboxaldehyde on pulmonary function and non-specific airway reactivity (methacholine-challenge) was also conducted on 4 rats/sex from the control and high exposure groups. The rats were monitored for signs of treatment-related alterations in respiratory parameters after exposure during the 9thexposure week and following the last day of exposure (at the end of the 13thweek of exposure). No treatment-related alterations in pulmonary function were observed during post-exposure monitoring. Prior to necropsy, the previously monitored rats were evaluated for exposure-related changes in nonspecific airway responsiveness (measured as a change in Penh) by exposure to increasing concentrations of aerosolized methacholine hydrochloride (MCh) in whole-body barometric plethysmography chambers. Compared to air-exposed control rats of the same sex, malebut not female rats exposed to 24.0 mg/m3had increased airway responsiveness to MCh challenge as evidenced by Penh values that were statistically higher than control rats. The observed increase in airway reactivity in only one sex and the absence of morphologic evidence of pulmonary airway inflammation or remodeling suggests exposure-related irritation, and not allergic sensitization, as the underlying cause of the increased responsiveness to methacholine challenge.

Histopathological lesions were limited to the anterior nasal cavity, consistent with the irritant qualities of 1,3- and 1,4-cyclohexanedicarboxaldehydeBased on the absence of exposure-related systemic effects, the 13-week no-observed-adverse-effect level (NOAEL) for 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors in male and female F344/DuCrl rats was determined to be greater than the highest concentration tested, 24.0 mg/m3. Based upon the localized, point of contact histopathological effects in the anterior nasal passage, the no-observed-effect level (NOAEL) for male and female F344/DuCrl rats repeatedly exposed to 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors for 13 weeks (65 exposures) was and 2.67 mg/m3.

Conclusions:
The adminsitration of the test material to male and female rats for 90-days (6h/d) resulted in local, site of contact irritation effects to the upper respiratory tract only. There was no indication of systemci toxicity at any dose level. The methacholine-challenge provided further information for the presence of an irritating effect (rather than an allergic reaction). The NOAEL for local effects is therefore the mid dose group - 2.67 mg/m3, and the NOAEL for systemic toxicity is the top dose of 24 mg/m3.
Executive summary:

This study was designed to evaluate the potential for local (portal-of-entry) and systemic toxicity from inhalation of1,3- and 1,4-cyclohexanedicarboxaldehyde. Groups of ten male and ten female F344/DuCrl rats were exposed six hours/day, five consecutive days/week for 13 weeks (a total of 65 exposures) using a flow-past nose-only inhalation exposure system. The rats were exposed to analytically-determined time-weighted average (TWA) concentrations of0,0.30 ± 0.20, 0, or 24.0 ± 5.2(study TWA±standard deviation)mg 1,3- and 1,4-cyclohexanedicarboxaldehyde vapor/m3air (0, 0.05, 0.47, or 4.2 ppm, respectively). In-life observations (including ophthalmology), functional tests, feed consumption, body weights/body weight gains, urinalysis, coagulation, hematology, clinical chemistry and organ weights were evaluated. Bronchoalveolar lavage (BAL) was performed on all exposure groups to assess exposure-related pulmonary inflammation and injury by measuring the types and numbers of inflammatory cells, the total protein concentration and lactate dehydrogenase (LDH) activity in the recovered lavage fluid. At the end of 13 weeks of exposure necropsy was conducted on all animals and a detailed histopathologic examination of the entire respiratory tract was performed by light microscopy to assess treatment-related portal of entry effects. In addition, a detailed histopathologic examination of all other tissues/organs was performed on the control- and high-exposure group rats to identify treatment-related systemic toxicity.

There were no exposure-related effects on body weights/body weight gains, feed consumption, clinical observations, functional tests,hematology, prothrombin time, clinical chemistry, urinalysis, or organ weights. Based on BAL analyses, male and female rats exposed to1,3- and 1,4-cyclohexanedicarboxaldehyde vaporsat concentrations up to and including 24.0 mg/m3showed no evidence of exposure-related cellular injury or inflammation. Compared to control rats, no increase in total BAL cell numbers and no evidence of acute neutrophilic inflammation were detected in any test material-exposed group. No treatment-related alterations were noted in BAL protein or LDH levels.

There were no treatment-related gross pathologic observations.

There was no histopathological evidence of treatment-related systemic toxicity in males or females exposed to the highest exposure concentration of 24.0 mg/m3. Males exposed to 2.67 or 24.0 mg/m3and females exposed to 24.0 mg/m3had treatment-related histopathological changes that were confined to the anterior nasal cavity consistent withlocalized, point of contact irritant effects resulting from repeated inhalation of 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors. The effects observed in the males at the 2.67 mg/m3 were very slight and represented an adaptive change (increase in mucouse secretion) rather than an adverse effect. Nasal airways of males exposed to 0.30 mg/m3and females exposed to 0.30 or 2.67 mg/m3had no treatment-related changes. There were no treatment-related histopathological changes in the posterior nasal airways, larynx, trachea or lungs in males or females in any of the exposure groups.

A limited assessment of the effects of repeated inhalation exposure to 1,3- and 1,4-cyclohexanedicarboxaldehyde on pulmonary function and non-specific airway reactivity (methacholine-challenge) was also conducted on 4 rats/sex from the control and high exposure groups. The rats were monitored for signs of treatment-related alterations in respiratory parameters after exposure during the 9thexposure week and following the last day of exposure (at the end of the 13thweek of exposure). No treatment-related alterations in pulmonary function were observed during post-exposure monitoring. Prior to necropsy, the previously monitored rats were evaluated for exposure-related changes in nonspecific airway responsiveness (measured as a change in Penh) by exposure to increasing concentrations of aerosolized methacholine hydrochloride (MCh) in whole-body barometric plethysmography chambers. Compared to air-exposed control rats of the same sex, malebut not female rats exposed to 24.0 mg/m3had increased airway responsiveness to MCh challenge as evidenced by Penh values that were statistically higher than control rats. The observed increase in airway reactivity in only one sex and the absence of morphologic evidence of pulmonary airway inflammation or remodeling suggests exposure-related irritation, and not allergic sensitization, as the underlying cause of the increased responsiveness to methacholine challenge.

Histopathological lesions were limited to the anterior nasal cavity, consistent with the irritant qualities of 1,3- and 1,4-cyclohexanedicarboxaldehydeBased on the absence of exposure-related systemic effects, the 13-week no-observed-adverse-effect level (NOAEL) for 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors in male and female F344/DuCrl rats was determined to be greater than the highest concentration tested, 24.0 mg/m3. Based upon the localized, point of contact histopathological effects in the anterior nasal passage, the no-observed-effect level (NOAEL) for male and female F344/DuCrl rats repeatedly exposed to 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors for 13 weeks (65 exposures) was and 2.67 mg/m3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
24 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
This is GLP and OECD 413 (USEPA OPPTS 870.3465, EEC PartB. 29) quideline study. Overall, the quality of the whole study is very good.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 July 2013-6 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was not conducted according to guidelines but was conducted according to GLPs and the report contains sufficient data for interpretation of study results.
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Qualifier:
according to
Guideline:
other: EEC, Part B.29
Principles of method if other than guideline:
This study was also designed to evaluate the potential for local (portal-of-entry) and systemic toxicity from inhalation of 1,3- and 1,4-cyclohexanedicarboxaldehyde.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: see "Details on inhalation exposure"
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
six hours/day, five consecutive days/week for 13 weeks.
No. of animals per sex per dose:
Male and female (each): 10 animals at 0 mg/m3
Male and female (each): 10 animals at 0.3 mg/m3
Male and female (each): 10animals at 2.67 mg/m3
Male and female (each): 10animals at 24.0 mg/m3
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
local signs of irritation in the respiratory tract
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEC
Remarks:
Systemic
Effect level:
> 24 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; mortality; body weight; food consumption; food efficiency; water consumption and compound intake; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology; other.
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
ca. 2.67 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based upon the localized, point of contact histopathological effects in the anterior nasal passage.
Dose descriptor:
NOEC
Remarks:
Local
Effect level:
ca. 2.67 mg/m³ air
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based upon the localized, point of contact histopathological effects in the anterior nasal passage.
Critical effects observed:
not specified

There were no exposure-related effects on body weights/body weight gains, feed consumption, clinical observations, functional tests,hematology, prothrombin time, clinical chemistry, urinalysis, or organ weights. Based on BAL analyses, male and female rats exposed to1,3- and 1,4-cyclohexanedicarboxaldehyde vaporsat concentrations up to and including 24.0 mg/m3showed no evidence of exposure-related cellular injury or inflammation. Compared to control rats, no increase in total BAL cell numbers and no evidence of acute neutrophilic inflammation were detected in any test material-exposed group. No treatment-related alterations were noted in BAL protein or LDH levels.

There were no treatment-related gross pathologic observations.

There was no histopathological evidence of treatment-related systemic toxicity in males or females exposed to the highest exposure concentration of 24.0 mg/m3. Males exposed to 2.67 or 24.0 mg/m3and females exposed to 24.0 mg/m3had treatment-related histopathological changes that were confined to the anterior nasal cavity consistent withlocalized, point of contact irritant effects resulting from repeated inhalation of 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors. The effects observed in the males at the 2.67 mg/m3 were very slight and represented an adaptive change (increase in mucouse secretion) rather than an adverse effect. Nasal airways of males exposed to 0.30 mg/m3and females exposed to 0.30 or 2.67 mg/m3had no treatment-related changes. There were no treatment-related histopathological changes in the posterior nasal airways, larynx, trachea or lungs in males or females in any of the exposure groups.

A limited assessment of the effects of repeated inhalation exposure to 1,3- and 1,4-cyclohexanedicarboxaldehyde on pulmonary function and non-specific airway reactivity (methacholine-challenge) was also conducted on 4 rats/sex from the control and high exposure groups. The rats were monitored for signs of treatment-related alterations in respiratory parameters after exposure during the 9thexposure week and following the last day of exposure (at the end of the 13thweek of exposure). No treatment-related alterations in pulmonary function were observed during post-exposure monitoring. Prior to necropsy, the previously monitored rats were evaluated for exposure-related changes in nonspecific airway responsiveness (measured as a change in Penh) by exposure to increasing concentrations of aerosolized methacholine hydrochloride (MCh) in whole-body barometric plethysmography chambers. Compared to air-exposed control rats of the same sex, malebut not female rats exposed to 24.0 mg/m3had increased airway responsiveness to MCh challenge as evidenced by Penh values that were statistically higher than control rats. The observed increase in airway reactivity in only one sex and the absence of morphologic evidence of pulmonary airway inflammation or remodeling suggests exposure-related irritation, and not allergic sensitization, as the underlying cause of the increased responsiveness to methacholine challenge.

Histopathological lesions were limited to the anterior nasal cavity, consistent with the irritant qualities of 1,3- and 1,4-cyclohexanedicarboxaldehydeBased on the absence of exposure-related systemic effects, the 13-week no-observed-adverse-effect level (NOAEL) for 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors in male and female F344/DuCrl rats was determined to be greater than the highest concentration tested, 24.0 mg/m3. Based upon the localized, point of contact histopathological effects in the anterior nasal passage, the no-observed-effect level (NOAEL) for male and female F344/DuCrl rats repeatedly exposed to 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors for 13 weeks (65 exposures) was and 2.67 mg/m3.

Conclusions:
The adminsitration of the test material to male and female rats for 90-days (6h/d) resulted in local, site of contact irritation effects to the upper respiratory tract only. There was no indication of systemci toxicity at any dose level. The methacholine-challenge provided further information for the presence of an irritating effect (rather than an allergic reaction). The NOAEL for local effects is therefore the mid dose group - 2.67 mg/m3, and the NOAEL for systemic toxicity is the top dose of 24 mg/m3.
Executive summary:

This study was designed to evaluate the potential for local (portal-of-entry) and systemic toxicity from inhalation of1,3- and 1,4-cyclohexanedicarboxaldehyde. Groups of ten male and ten female F344/DuCrl rats were exposed six hours/day, five consecutive days/week for 13 weeks (a total of 65 exposures) using a flow-past nose-only inhalation exposure system. The rats were exposed to analytically-determined time-weighted average (TWA) concentrations of0,0.30 ± 0.20, 0, or 24.0 ± 5.2(study TWA±standard deviation)mg 1,3- and 1,4-cyclohexanedicarboxaldehyde vapor/m3air (0, 0.05, 0.47, or 4.2 ppm, respectively). In-life observations (including ophthalmology), functional tests, feed consumption, body weights/body weight gains, urinalysis, coagulation, hematology, clinical chemistry and organ weights were evaluated. Bronchoalveolar lavage (BAL) was performed on all exposure groups to assess exposure-related pulmonary inflammation and injury by measuring the types and numbers of inflammatory cells, the total protein concentration and lactate dehydrogenase (LDH) activity in the recovered lavage fluid. At the end of 13 weeks of exposure necropsy was conducted on all animals and a detailed histopathologic examination of the entire respiratory tract was performed by light microscopy to assess treatment-related portal of entry effects. In addition, a detailed histopathologic examination of all other tissues/organs was performed on the control- and high-exposure group rats to identify treatment-related systemic toxicity.

There were no exposure-related effects on body weights/body weight gains, feed consumption, clinical observations, functional tests,hematology, prothrombin time, clinical chemistry, urinalysis, or organ weights. Based on BAL analyses, male and female rats exposed to1,3- and 1,4-cyclohexanedicarboxaldehyde vaporsat concentrations up to and including 24.0 mg/m3showed no evidence of exposure-related cellular injury or inflammation. Compared to control rats, no increase in total BAL cell numbers and no evidence of acute neutrophilic inflammation were detected in any test material-exposed group. No treatment-related alterations were noted in BAL protein or LDH levels.

There were no treatment-related gross pathologic observations.

There was no histopathological evidence of treatment-related systemic toxicity in males or females exposed to the highest exposure concentration of 24.0 mg/m3. Males exposed to 2.67 or 24.0 mg/m3and females exposed to 24.0 mg/m3had treatment-related histopathological changes that were confined to the anterior nasal cavity consistent withlocalized, point of contact irritant effects resulting from repeated inhalation of 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors. The effects observed in the males at the 2.67 mg/m3 were very slight and represented an adaptive change (increase in mucouse secretion) rather than an adverse effect. Nasal airways of males exposed to 0.30 mg/m3and females exposed to 0.30 or 2.67 mg/m3had no treatment-related changes. There were no treatment-related histopathological changes in the posterior nasal airways, larynx, trachea or lungs in males or females in any of the exposure groups.

A limited assessment of the effects of repeated inhalation exposure to 1,3- and 1,4-cyclohexanedicarboxaldehyde on pulmonary function and non-specific airway reactivity (methacholine-challenge) was also conducted on 4 rats/sex from the control and high exposure groups. The rats were monitored for signs of treatment-related alterations in respiratory parameters after exposure during the 9thexposure week and following the last day of exposure (at the end of the 13thweek of exposure). No treatment-related alterations in pulmonary function were observed during post-exposure monitoring. Prior to necropsy, the previously monitored rats were evaluated for exposure-related changes in nonspecific airway responsiveness (measured as a change in Penh) by exposure to increasing concentrations of aerosolized methacholine hydrochloride (MCh) in whole-body barometric plethysmography chambers. Compared to air-exposed control rats of the same sex, malebut not female rats exposed to 24.0 mg/m3had increased airway responsiveness to MCh challenge as evidenced by Penh values that were statistically higher than control rats. The observed increase in airway reactivity in only one sex and the absence of morphologic evidence of pulmonary airway inflammation or remodeling suggests exposure-related irritation, and not allergic sensitization, as the underlying cause of the increased responsiveness to methacholine challenge.

Histopathological lesions were limited to the anterior nasal cavity, consistent with the irritant qualities of 1,3- and 1,4-cyclohexanedicarboxaldehydeBased on the absence of exposure-related systemic effects, the 13-week no-observed-adverse-effect level (NOAEL) for 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors in male and female F344/DuCrl rats was determined to be greater than the highest concentration tested, 24.0 mg/m3. Based upon the localized, point of contact histopathological effects in the anterior nasal passage, the no-observed-effect level (NOAEL) for male and female F344/DuCrl rats repeatedly exposed to 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors for 13 weeks (65 exposures) was and 2.67 mg/m3.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
2.67 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
A 90d study is available and sufficient to characterise the local effects of this substance on the respiratory tract.

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

Repeated dose oral toxicity data:

14 -day range findings study (for OECD 421)

This study was conducted as a range finder for an OECD 421 reproductive screening study. The test substance was administered in corn oil via gavage to Wistar rats at an equivolume of 4 mL/kg bw/day at the dose levels of 250, 500 and 1000 mg/kg bw/day. Concurrently, vehicle control group animals were administered the vehicle alone (corn oil) at the same dose volume. Each group in the study was comprised of 3 rats per sex.

All the rats in the study were observed for clinical signs twice on the first day of treatment. Subsequently, the observation was carried out thrice daily until the end of treatment. The rats were observed for morbidity and mortality twice daily (once daily on weekends). Individual body weights were recorded prior to test substance administration for all rats on Day 1 and on Days 4, 7, 11 and 14. Additionally, body weight was also measured on Day 15 following overnight fasting. Feed consumption was measured on Day 4 (representing the feed consumption between Days 1-4) and on Days 7, 11 and 14 (representing the feed consumption between Days 4-7, Days 7-11 and Days 11-14, respectively) for each rat. Kidney and liver weights were recorded from all the terminally sacrificed rats after gross pathology examination.

The daily oral gavage of 1,3 and 1,4 Cyclohexanecarboxaldehyde for 14 consecutive days at the dose of 250 mg/kg body weight/day had no test substance-related effects on the general health of the animals, body weights, net body weight gains, feed consumption, organ weights of the liver and kidneys and gross pathology. However, at 1000 mg/kg bw/day, treatment related mortalities were observed (2/6), and one male rat was euthanized moribund. Clinical signs of hypo activity and salivation were observed at 500 and 1000 mg/kg bw/day. The mean and net body weight gains and feed consumption were lower at 1000 mg/kg bw/day compared to the concurrent control group. Percent body weight gain was also decreased in males (32%) and females (20%) at 500 mg/kg bw/day. The treatment resulted in an increase in the absolute and relative liver weights in females at 1000 mg/kg bw/day. There was also a marginal increase in absolute (15%) and relative (16%) liver weights in females at 500 mg/kg bw/day. Gross pathological findings included erosions and thickening of the stomach and intestines of some animals in the 1000 mg/kg bw/day group. Raised foci were present in the stomach of 3 males and 2 females in the 500 mg/kg bw/day group.

The results of this range-finding study included body weight effects as well as point of contact irritation in the stomach at 500 mg/kg bw/day, and death and moribundity at 1000 mg/kg bw/day. Based on the absence of findings in the low dose group, the NOEL for local toxicity was considered to be 250 mg/kg bw/day. The study did not provide sufficient information to derive a NOAEL for systemic toxicity, however the observations in the mid and high dose group appear to be completely attributable to the highly irritant nature of the test material and as such the NOAEL for systemic toxicity is potentially the mid or top dose.

OECD 421 study (Study details reported in section 7.8.1).

This study represents the longest available oral repeated dose study for the test substance. The test substance was administered in graduated doses to three groups of male and female rats. The males were dosed for a period of at least six weeks (which included 2 weeks prior mating, 2 weeks during mating and 2 weeks post mating), up to and including the day before sacrifice. Females were dosed throughout the treatment period. This included two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and four days after delivery.

The treatment-related clinical sign of slight salivation was observed during and after gavage administration in the 400 mg/kg bw/day dose group males (9/10) and females (2/10). This persisted for 10-15 minutes post dosing and rats returned to normal thereafter. All adult animals survived until termination. Treatment with the test substance 1,3 and 1,4 Cyclohexanecarboxaldehyde did not cause any test substance related effects on general health, body weights, feed consumption, mating and fertility, mean number of corpora lutea and implantations, live birth index, mean litter size, growth and development of pups. There were no test substance related changes in terminal fasting body weights and organ weights at all the doses tested.

Multiple raised foci and/or diffuse thickening was observed grossly in non glandular stomach in 400 mg/kg/day dose males and females. Microscopically these observations were associated with ulcers and epithelial hyperplasia/hyperkeratosis and were considered test substance-related adverse changes. The NOAEL for local effects was determined to be 150 mg/kg bw/day, while the NOAEL for systemic effects was 400 mg/kg bw/day.

 

28-day oral Gavage study

The test substance was administered daily for 28 days by oral gavage to SPF bred Wistar rats. The dose levels chosen were 150, 450 and 1000 mg/kg bw/day, and test substance was administered undiluted. The control group received water. The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; water consumption between days 21-25; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Treatment resulted in a marked irritant effect on the stomach down to the lowest administered dose of 150 mg/kg/day. The primary and most severe finding was forestomach ulceration with submucosal inflammation in the stomach which was recorded in a dose related fashion in all dose groups, reaching very severe grade at 1000 mg/kg/day. These findings correlated to necropsy findings. Ulceration of the forestomach was the cause of moribundity and subsequent sacrifice of one male at 1000 mg/kg/day. Clinical signs and deviations in clinical pathology parameters observed at 450 and 1000 mg/kg/day as well as lower body weights and food intake at 1000 mg/kg/day were also considered to have resulted from the stomach effects noted at these dose levels. Increased adrenal weights and reduced thymus weights at 1000 mg/kg/day were considered to be a secondary non-specific stress response. Similarly, the alterations noted in the mesenteric lymph node recorded variously at 450 and 1000 mg/kg/day as well as higher white blood cell counts at 1000 mg/kg/day may be regarded as secondary reactive responses to the inflammation and necrosis in the stomach.

Based on the stomach effects observed at dose levels down to 150 mg/kg/day, a No Observed Adverse Effect Level (NOAEL) for local toxicity could not be established. All other findings were either considered to have resulted secondarily from the stomach effects or were considered not to constitute an adverse effect. Therefore, a NOAEL of 1000 mg/kg/day for systemic toxicity was established.

Summary of oral toxicity findings

From the available repeated dose oral studies it is clear that this substance is highly irritating and that local effects in the gastrointestinal tract (in particular the stomach) drive the No and Low effect levels. The degree of local irritation is dependant on the dose regimen used and this is evident when comparing the OECD 421 and 14 day range finder (both of which used a vehicle) with the 28-day study (undiluted test material). In the former, there was an apparent no effect level for local effects of 150 mg/kg bw, whereas in the latter, even the low dose of 150 mg/kg bw produced some evidence of local irritation.

Considering the potential systemic toxicity, there is little to no evidence of systemic toxicity with all findings appearing to be secondary to the irritation or the stress produced by the irritation of the test material. As a consequence the top dose level of 1000 mg/kg bw/day in the 28 day study was considered to be a NOAEL for systemic toxicity.

 

Repeated dose Inhalation data

14-day range finding inhalation study

In preparation for a 13-week inhalation toxicity study in rats, a range finding study (14 days) was performed to evaluate the potential for local (portal-of-entry) and systemic toxicity from inhalation of 1,3- and 1,4-cyclohexanedicarboxaldehyde and to aid in setting exposure levels for the subsequent 13-week subchronic inhalation study. 

Initially, groups of 5 male and 5 female F344/DuCrl rats were exposed to dose levels of 0, 43.4, 426.1, or 1102.8 mg 1,3- and 1,4-cyclohexanedicarboxaldehyde/m3air 6 hours/day, 5 days/week for 2 weeks. However due to significant adverse clinical effects the high and mid dose animals were removed from exposures on day 4 (following 2 and 3 days of exposure, respectively) and humanely euthanized. Limited data were collected from the 43.4 mg/m3group, and were included in the results for the repeated range finding study.

In the repeated range finding studygroups of 5 male and 5 female F344/DuCrl rats were exposed six hours/day, five consecutive days/week for 2 weeks (a total of 10 exposures) to1,3- and 1,4-cyclohexanedicarboxaldehydevapors using a flow-past nose-only inhalation exposure system. The rats were exposed to time-weighted average (TWA) concentrations of 0, 4.4 ± 3.2, 26.8 ± 9.4, or 93.2 ± 11.3 mg 1,3- and 1,4-cyclohexanedicarboxaldehyde vapor/m3air (study mean±standard deviation), or 0, 0.8, 4.7, or 16.3 ppm, respectively. 

Bodyweight gain and terminal bodyweight in the high dose group (93.2 mg/m3) were statistically identified as decreased from their respective controls when male and female data were combined. However when considered separately, mean terminal body weights were decreased in males (relative to control males) for all exposure groups in an exposure-dependent manner, whereas mean terminal body weights of female rats from all exposure groups were similar to the controls. Mean feed consumption values for male rats exposed to 93.2 mg/m3were statistically identified as decreased relative to control rats during the second week of the study.

There were no treatment-related changes in hematology or prothrombin time. The high dose group (male and female) had statistically significant decreases in alkaline phosphatase in both sexes compared to controls. The high dose group also had statistically significant treatment-related decrements in absolute thymic weight relative to controls.

Based on bronchiole alveolar lavage (BAL) analyses, high dose male and female rats had no discernible signs of exposure-related cellular injury or inflammation.

Treatment-related gross lesions were restricted to females exposed to 93.2 mg/m3. Two animals had soiling of the external nares, one of which also had a decreased amount of fat. The single finding of decreased abdominal fat correlated with a decreased terminal body weight in this animal and was corroborated histologically as atrophy of mesenteric fat and interpreted to be treatment-related.

Lesions of the respiratory tract occurred inthe anterior nasal cavity of animals exposed to 93.2 and 26.8 mg/m3and cranial larynx of animals exposed to 93.2 mg/m3. The nasal cavity of male and female rats exposed to 26.8 or 93.2 mg/m3had erosive/ulcerative, metaplastic/hyperplastic, and inflammatory changes to the squamous, respiratory, transitional epithelia, and hypertrophy of mucous cells lining the nasopharynx. The cranial larynx of male and female rats exposed to93.1 mg/m3had very slight squamous metaplasia of the surface epithelium overlying the cartilage at the base of the epiglottis.

A subgroup of control and93.1 mg/m3exposure group rats were challenged with methacholine following 5 and 10 exposures to provide some assessment of non-specific airway reactivity. Although the 93.1 mg/m3exposure animals demonstrated increases in penh compared with the controls (males and females after 5 exposures; males only after 10 exposures), the overall magnitude of the response was decreased with increasing number of exposures, which suggests an adaptive response to repeated exposure to a respiratory irritant.

With the exception of thymic weight decrements (indicative of a stress response) and changes occurring secondary to body weight effects, systemic pathology was not observed in animals exposed to 93.2 mg/m31,3- and 1,4-cyclohexanedicarboxaldehyde. Histopathological lesions were limited to the upper respiratory tract, consistent with the irritant qualities of 1,3- and 1,4-cyclohexanedicarboxaldehyde. Decreased feed-consumption (statistically identified in males), statistically identified decreased in-life body weights (male and female combined), and decreased terminal body weights (males) were interpreted as treatment-related body weight effects occurring secondary to stress and histological lesions of the upper respiratory tract. Based on the absence of local effects, 4.4 mg/m3was determined to be the 2-week no observed effect concentration (NOEC) for vapors of 1,3- and 1,4-cyclohexanedicarboxaldehyde in F344/DuCrl rats. For systemic effects, it is probable that the top dose of 93.2 mg/m3represents the NOAEL.

13 week inhalation study

This study was designed to evaluate the potential for local (portal-of-entry) and systemic toxicity from inhalation of 1,3- and 1,4-cyclohexanedicarboxaldehyde. Groups of ten male and ten female F344/DuCrl rats were exposed six hours/day, five consecutive days/week for 13 weeks (a total of 65 exposures) using a flow-past nose-only inhalation exposure system. The rats were exposed to analytically-determined time-weighted average (TWA) concentrations of 0,0.30 ± 0.20, 0, or 24.0 ± 5.2 (study TWA±standard deviation) mg 1,3- and 1,4-cyclohexanedicarboxaldehyde vapor/m3air (0, 0.05, 0.47, or 4.2 ppm, respectively). In-life observations (including ophthalmology), functional tests, feed consumption, body weights/body weight gains, urinalysis, coagulation, hematology, clinical chemistry and organ weights were evaluated. Bronchoalveolar lavage (BAL) was performed on all exposure groups to assess exposure-related pulmonary inflammation and injury by measuring the types and numbers of inflammatory cells, the total protein concentration and lactate dehydrogenase (LDH) activity in the recovered lavage fluid. At the end of 13 weeks of exposure necropsy was conducted on all animals and a detailed histopathologic examination of the entire respiratory tract was performed by light microscopy to assess treatment-related portal of entry effects. In addition, a detailed histopathologic examination of all other tissues/organs was performed on the control- and high-exposure group rats to identify treatment-related systemic toxicity.

There were no exposure-related effects on body weights/body weight gains, feed consumption, clinical observations, functional tests,hematology, prothrombin time, clinical chemistry, urinalysis, or organ weights. Based on BAL analyses, male and female rats exposed to1,3- and 1,4-cyclohexanedicarboxaldehyde vaporsat concentrations up to and including 24.0 mg/m3showed no evidence of exposure-related cellular injury or inflammation. Compared to control rats, no increase in total BAL cell numbers and no evidence of acute neutrophilic inflammation were detected in any test material-exposed group. No treatment-related alterations were noted in BAL protein or LDH levels.

There were no treatment-related gross pathologic observations.

There was no histopathological evidence of treatment-related systemic toxicity in males or females exposed to the highest exposure concentration of 24.0 mg/m3. Males exposed to 2.67 or 24.0 mg/m3and females exposed to 24.0 mg/m3had treatment-related histopathological changes that were confined to the anterior nasal cavity consistent withlocalized, point of contact irritant effects resulting from repeated inhalation of 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors. The effects observed in the males at the 2.67 mg/m3 were very slight and represented an adaptive change (increase in mucouse secretion) rather than an adverse effect. Nasal airways of males exposed to 0.30 mg/m3and females exposed to 0.30 or 2.67 mg/m3had no treatment-related changes. There were no treatment-related histopathological changes in the posterior nasal airways, larynx, trachea or lungs in males or females in any of the exposure groups.

A limited assessment of the effects of repeated inhalation exposure to 1,3- and 1,4-cyclohexanedicarboxaldehyde on pulmonary function and non-specific airway reactivity (methacholine-challenge) was also conducted on 4 rats/sex from the control and high exposure groups. The rats were monitored for signs of treatment-related alterations in respiratory parameters after exposure during the 9thexposure week and following the last day of exposure (at the end of the 13thweek of exposure). No treatment-related alterations in pulmonary function were observed during post-exposure monitoring. Prior to necropsy, the previously monitored rats were evaluated for exposure-related changes in nonspecific airway responsiveness (measured as a change in Penh) by exposure to increasing concentrations of aerosolized methacholine hydrochloride (MCh) in whole-body barometric plethysmography chambers. Compared to air-exposed control rats of the same sex, malebut not female rats exposed to 24.0 mg/m3had increased airway responsiveness to MCh challenge as evidenced by Penh values that were statistically higher than control rats. The observed increase in airway reactivity in only one sex and the absence of morphologic evidence of pulmonary airway inflammation or remodeling suggests exposure-related irritation, and not allergic sensitization, as the underlying cause of the increased responsiveness to methacholine challenge.

Histopathological lesions were limited to the anterior nasal cavity, consistent with the irritant qualities of 1,3- and 1,4-cyclohexanedicarboxaldehydeBased on the absence of exposure-related systemic effects, the 13-week no-observed-adverse-effect level (NOAEL) for 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors in male and female F344/DuCrl rats was determined to be greater than the highest concentration tested, 24.0 mg/m3. Based upon the localized, point of contact histopathological effects in the anterior nasal passage, the no-observed-effect level (NOAEL) for male and female F344/DuCrl rats repeatedly exposed to 1,3- and 1,4-cyclohexanedicarboxaldehyde vapors for 13 weeks (65 exposures) was and 2.67 mg/m3.

Summary of inhalation findings

As with the oral studies, it is apparent that there is little to no systemic toxicity and that the key effect is upper respiratory tract irritation. The NOEL for this local effect is 2.67 mg/m3and this will serve as the point of departure for the risk assessment of this substance.

 

No repeated dose dermal toxicity data are available.

A note on purity:

The registered substance has a purified and a crude grade, the latter containing heptane as the most significant impurity (up to 7%). The data available were generated using the purified grade but are considered sufficient to characterise both purified and crude grades. The toxicity of heptane is well characterised and will not contribute to the toxicity of this substance. Heptane is not a reproductive toxicant, has a low order of repeated dose toxicity and is not genotoxic/carcinogenic. The other impurities present within the crude grade are structurally similar to the main constituent (mono aldehydes) and as such are expected to have similar toxicity to the main constituents (irritancy, sensitisng potential, absence of genotoxicity).

A note on the conduct of a 90 -day study without submitting a test proposal:

The 90 -day study was performed in order to support a Tier II notification in China. The timeline for this notification is late 2014 and therefore the 90 -day study was performed without submitting a test proposal in accordance with the REACH regulation. Prior to perfoming the study we confirmed that no 90 -day repeated dose toxicity studies were available (published) and we are not aware of any other manufacturers of the substance that could have provided such a study. As such we are confident that we have repeated an allready existing study.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Repeated dose study assigned a Klimisch 1 rating. This study represents the longest available oral repeated dose study for the test substance.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Repeated dose study assigned a Klimisch 1 rating. This study represents the longest available repeated inhalation dose study for the test substance.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Repeated dose study assigned a Klimisch 1 rating. This study represents the longest available repeated inhalation dose study for the test substance.

Justification for classification or non-classification

No systemic effects were detected in the available studies and no specific target organ toxicity classification is warranted. The substance appears to be irritating to the respiratory tract and this is addressed by a classification for respiratory tract irritation. (STOT SE cat 3)