1,3- AND 1,4-CYCLOHEXANDICARBOXYALDEHYDE

Endpoint summary

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

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

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Qualifier:

according to guideline

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. QA statement)

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 9^thexposure week and following the last day of exposure (at the end of the 13^thweek 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-cyclohexanedicarboxaldehyde. Based 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 9^thexposure week and following the last day of exposure (at the end of the 13^thweek 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-cyclohexanedicarboxaldehyde. Based 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

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Qualifier:

according to guideline

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. QA statement)

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 9^thexposure week and following the last day of exposure (at the end of the 13^thweek 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-cyclohexanedicarboxaldehyde. Based 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 9^thexposure week and following the last day of exposure (at the end of the 13^thweek 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-cyclohexanedicarboxaldehyde. Based 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 9^thexposure week and following the last day of exposure (at the end of the 13^thweek 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-cyclohexanedicarboxaldehyde. Based 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)