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Toxicological information

Toxicity to reproduction: other studies

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Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Assessment of CoA conjugate formation using plated rat hepatocytes.
GLP compliance:
no
Type of method:
in vitro

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
no data available
Specific details on test material used for the study:
Further materials tested:
3-(3-tert-butylphenyl)-2-methylpropanal (meta- Lysmeral);
3-(4-tert-butylphenyl)-2-methylpropanoic acid (Lysmerylic acid);
3-(p-tert-butylphenyl)-2-methylpropanol (Lysmerol);
3-(4-tert-butylphenyl)-propanal (BHCA)
3-(4-isopropylphenyl)-2-methylpropanal (PMHCA)
3-(4-isopropylphenyl)-propanal (PHCA)
3-(3-isopropylphenyl)-3-methylpropanal (miP2MHCA)
3-(4-isobutylphenyl)-2-methylpropanal (iBMHCA)
p-tert-butyltoluene (TBT)
p-tert-butylbenzoic acid (TBBA)
p-isopropylbenzoic acid
3-(4-ethylphenyl)-2,2-dimethylpropanal (Floralozone)
alpha-methyl-1,3-benzodioxole-5-propanal (Tropional)
3-(4-methoxyphenyl)-2-methylpropanal (Fennaldehyde)
3-(4-tolyl)propanal (Jasmorange)
3-(4-isobutyl-2-methylphenyl)propanal (Nympheal)
Benzoic acid
p-Hydroxy-benzoic acid
ethyl 4-hydroxybenzoate (Ethylparaben)

Results and discussion

Any other information on results incl. tables

p-Alkyl benzoyl-Coenzyme A formation from TBBA or Lysmeral in plated rat hepatocytes

TBBA is rapidly transformed top-tert-butyl-benzoyl-CoA and accumulates to stable levels within 0.5 – 4 h (see Figure 2 in the document attached)

Saturation observed at test concentrations (5,10,50 µM).

After Lysmeral addition, TBBA is formed rapidly and is conjugated to CoA.

Concentration of the TBBA-CoA conjugate remains stable over time indicating that CoA conjugated benzoic acid is not rapidly and/or quantitatively transferred to secondary acceptors such as glycine or taurine.

TBBA-CoA conjugate concentrations (1-2µM) are higher compared to endogenous oleoyl-CoA, palmitoyl-CoA, arachidonoyl-CoA (<0.1 µM) -> Accumulation of the TBBA-CoA conjugates in treated cells may competitively inhibit other CoA dependent cellular processes.

 

Formation of benzoyl-Coenzyme A conjugates for Lysmeral-like materials and correlation to rat male reproductive toxicity

Similar accumulation (sustained accumulation for 22 h incubation) of the corresponding alkyl-benzoyl-CoA conjugate was observed for a number of chemicals with a para-substituent at the benzyl ring (BMHCA-acid, BMHCA-alcohol, PMHCA, PHCA, iBMHCA, p-tert-butyltoluene, p-isopropyl benzoic acid). They alsoexhibit male reprotoxic effects in the rat.

No accumulation of the corresponding alkyl benzoyl-CoA conjugate (i.e. especially low levels at the 22 h time point) was observed for m-BMHCA, m-iP2MHCA, Floralozone, Tropional, Fennaldehyde, Jasmorange, NymphealTM, benzoic acid, p-hydroxy benzoic acid and ethylparaben. No reprotoxic effects were detected for these chemicals in the rat in vivo.

Table: Structures, adverse reprotoxic effects on male rats and accumulation of benzoyl-CoA conjugates in plated rat hepatocytes.All chemicals were tested at 50 µM and selected chemicals were also tested at 5 µM in plated rat hepatocytes. Benzoyl-CoA conjugates refer to the CoA-conjugate formed from the benzoic acid derived from the test chemical and conjugated to CoA. Benzoyl-CoA conjugates were quantified versus a synthetic sample of TBBA-CoA using LC-HRMS analysis. Data for individual repetitions are expressed as % versus the formation of TBBA-CoA from Lysmeral at the given time point and at the corresponding test chemical concentration. Lysmeral was tested in all experiments as reference. bd, below detection level; nd, not determined.*only dose tested.

Name

CAS Nr.

Structure

LOAEL for male reprotoxic effects in rat

Benzoyl-CoA-conjugate

 (50 µM)

Benzoyl-CoA-conjugate

(5 µM)

 

 

 

(mg/kg bw/day)

4 h

22 h

4h

22h

3-(4-tert-butylphenyl)-2-methylpropanal (Lysmeral)

80-54-6

50

(Givaudan 1986A)

100

100

100

100

3-(3-tert-butylphenyl)-2-methylpropanal (meta- Lysmeral)

62518-65-4

> 450

(BASF SE 2011A)

18

24

8

13

3-(4-tert-butylphenyl)-2-methylpropanoic acid (Lysmerylic acid)

66735-04-4

50*

(BASF SE 2006A)

90

94

90

79

3-(p-tert-butylphenyl)-2-methylpropanol (Lysmerol)

56107-04-1

50

(BASF SE 2011B)

62

123

119

67

3-(4-tert-butylphenyl)-propanal (BHCA)

18127-01-0

25 (100)

(RIFM 2016, ECHA 2017F)

115

143

95

102

3-(4-isopropylphenyl)-2-methylpropanal (PMHCA)

103-95-7

75

(ECHA 2017A)

64

66

59

82

3-(4-isopropylphenyl)-propanal (PHCA)

7775-00-0

75

(RIFM 2016)

96

81

90

42

3-(3-isopropylphenyl)-3-methylpropanal (m-iP2MHCA)

125109-85-5

> 250

(ECHA 2017B)

2

2

nd

nd

3-(4-isobutylphenyl)-2-methylpropanal (iBMHCA)

6658-48-6

25

(RIFM 2016)

53

44

24

17

p-tert-butyltoluene (TBT)

98-51-1

15

(Furuhashi 2007B)

127

116

nd

nd

p-tert-butylbenzoic acid (TBBA)

98-73-7

6 – 8

(Hunter 1965)

110

98

108

84

p-isopropylbenzoic acid

536-66-3

15

(Givaudan 2011)

77

69

56

35

Floralozone (3-(4-ethylphenyl)-2,2-dimethylpropanal)

67634-15-5

> 250

(RIFM 2016)

1

bd

nd

nd

Tropional (a-methyl-1,3-benzodioxole-5-propanal)

1205-17-0

> 1000*

(BASF SE 2010B)

9

10

nd

nd

Fennaldehyde (3-(4-methoxyphenyl)-2-methylpropanal)

5462-06-6

> 1000*

(BASF SE 2011B)

12

16

nd

nd

Jasmorange (3-(4-tolyl)propanal)

41496-43-9

> 1000*

(BASF SE 2011B)

6

3

nd

nd

NymphealTM

(3-(4-isobutyl-2-methylphenyl)propanal

 

> 500

(Laue 2017)

bd

bd

nd

nd

Benzoic acid

65-85-0

> 900

(ECHA 2017C)

1

bd

nd

nd

p-Hydroxy-benzoic acid

99-96-7

> 1000

(ECHA 2017D)

bd

bd

nd

nd

Ethylparaben (ethyl 4-hydroxybenzoate)

120-47-8

> 1043

(ECHA 2017E)

1

bd

nd

nd

 

Coenzyme A conjugate formation from other xenobiotic acids in vitro

Only para-alkyl benzoic acids with the alkyl substituent being at least an ethyl group, a sustained accumulation of the CoA conjugate was observed.

The carboxylic acid CoA conjugate of Lysmerylic acid is only transiently formed at low levels at early time points and not detectable at 22 h incubation in contrast to TBBA-CoA.

TBBA-glycine conjugate was found in plated hepatocytes when incubated with benzoic acid but not with Lysmeral or TBBA.

 

Comparison of p-alkyl benzoyl-Coenzyme A formation from Lysmeral in rat vs. human plateable hepatocytes indicates species specificity

Around 5-times lower TBBA-CoA levels were detected after 0.5 h incubation with Lysmeral in plated human versus rat hepatocytes (see Figure 3 in the document attached).

While stable levels of TBBA-CoA are formed in plated rat hepatocytes, a strong decrease over time is observed in human hepatocytes.

Comparable results were observed with TBBA as test chemical (see Figure 4 in the document attached).

In human hepatocytes, amounts and kinetics of TBBA-CoA and Lysmerylic acid-CoA during Lysmeral incubation were almost identical (i.e. rapid decrease and no accumulation), whereas different amounts of these two CoA conjugates were detected in rat hepatocytes (see Figure 5 in the document attached).

In human hepatocytes, kinetics of TBBA-CoA formation during Lysmeral or TBBA treatment are similar to those observed for a number of non-reprotoxic chemicals in rat hepatocytes such as e.g. m-BMHCA, Fennaldehyde (see Figure 6 in the document attached), Tropional and Jasmorange.

 

Intrinsic formation of an endogenous CoA conjugate as marker for cell metabolic capacity

Octanoyl-CoA is the most predominant CoA-conjugate formed in control cells and it is formed with a similar kinetic in plated rat and human hepatocytes (see Figure 7 in the document attached). -> The decrease of TBBA-CoA in human hepatocytes at 22h is not due to a human hepatocyte specific loss in the ability of CoA-conjugation over time.

Applicant's summary and conclusion

Conclusions:
The formation of p-alkyl-benzoyl-CoA conjugates for Lysmeral, TBBA and related molecules is identified in plated rat hepatocytes. The kinetics of this formation (sustained, high level of these metabolites) is a unique feature for p-alkyl-benzoic acids and their precursors and is not observed for other xenobiotic acids, which only form transient and lower levels of CoA conjugates.
The strong correlation between p-alkyl-benzoyl-CoA conjugate formation in rat hepatocytes by a well-defined group of chemicals and their reprotoxic effects in the male rat indicate that this metabolic fate is a critical hallmark for the reproductive toxicity outcome. Importantly, small modifications in the target molecule can eliminate both this metabolic outcome and the reproductive toxicity effects (e.g. 3-(4-isobutyl-2-methylphenyl)propanal).
Studies in human hepatocytes indicate, that a completely different metabolic outcome in relation to p-alkyl-benzoyl-CoA conjugates is observed. Lower absolute amounts of p-alkyl-benzoyl-CoA conjugates were detected. Additionally, the rapid decrease of the p-alkyl-benzoyl-CoA conjugates over time indicate a transient formation of these conjugates in contrast to their sustained accumulation at relatively high concentration in rat hepatocytes. Finally, the kinetics of CoA-ester formation in rat hepatocytes of non-reprotoxic derivatives resembles the kinetics seen for Lysmeral in human hepatocytes. These results strongly indicate that the observed metabolic fate of Lysmeral is rat-specific and suggests that the observed toxicity is a species-specific effect.